[Objective] Siberian Roe Deer (Capreolus pygargus) is a kind of important wildanimal with edible value, meidicinal and ornamental value for scientific research. To understand the biological characteristics of Siberian Roe Deer fibroblasts, trachea, lung and ear tissues were collected for cell culture. [Methods] In this study, wild male and female Siberian Roe Deers were collected as experimental materials from Daqingshan, Inner Mongolia Autonomous Region, China. The primary fibroblasts of trachea, lung and ear were established by tissue block monolayer culture method. The cell adherence rate, survival rate before freezing and after resuscitation, growth curve of cells from different tissue sources were explored, and the karyotype and the G-band characteristics were analyzed. Unless otherwise noted, all analyses were performed in at least triplicate. Student’s t-test was uses to calculate difference using Graphpad (Version 5.0) [Results] The experimental results showed that spindle-shaped or triangular cells were newly grown along the tissue blocks in the trachea and ear tissues of male roe deer after 2﹣4 days of culture. After 8﹣10 days of culture, the convergence degree of cells in the flask reached 60%, and after 11﹣12 days of culture, the convergence degree of cells in the flask reached 80% (Fig. 1). The morphology and growth of fibroblasts from trachea, ear and lung of the femalewere basically the same as those of the male (Fig. 2). The most densely populated fibroblasts from the trachea, ears and lungs were observed at P20, P12 and P9, respectively (Fig. 3). The adherence rates of fibroblasts from the three tissues increased gradually with time, and grew rapidly within 6 - 12 h. After 24 h, the adherence rates of fibroblasts from the trachea and ears were over 95%. The adherence rate of P3﹣P9 lung fibroblasts varied greatly, and the adherence rate reached more than 90% when the culture time was 24 h (Table 1). There was almost no difference in the cell survival rate of airway fibroblasts in different generations before cryopreservation, and the survival rates were all above 85%. There was little difference in cell survival rate between ear and lung fibroblasts in different generations before cryopreservation, and the survival rate was above 90%. The recovery rate of fibroblasts after freezing gradually decreased with the increase of cell passage times (Table 2). The growth curves of fibroblasts from the trachea, ears and lungs show a typical “S” shape (Table 3, Fig. 4). Mycoplasma test results were negative (Fig. 5). The results of H.E staining showed that the three kinds of fibroblasts grew in a spiral shape, with light blue-purple nuclei, light red cytoplasm and that long spindle-shaped or triangular cellswere typical fibroblasts in morphology (Fig. 6). Karyotype and G-banding analysis showed that the number of chromosomes in male deer fibroblasts was 2n = 70 and Bs = 5, among which there were 34 pairs of autosomes, with the morphological type of 12 submetacentric + 22 submetacentric, one pair of sex chromosomes, with the morphological type of X being metacentric, Y being st and five B chromosomes (Fig. 7a, Fig. 8a, Table 4). The number of chromosomes in female fibroblastswas 2n = 70 and Bs = 8, among which there were 34 pairs of autosomes, with morphological type was 29 submetacentric + 5 subacrocentric, one pair of sex chromosome with morphological type of X being submetacentric, and eight B chromosomes (Fig. 7b, Fig. 8b, Table 5). [Conclusion] In this study, three tissue-derived fibroblast cell lines from male and female roe deer were successfully established. When cultured in vitro, they grew well and maintained the stability of genetic stability. The karyotypes and G-banding altas of male and female roe deer chromosomes were drawn. This study provides material and basic technical support for further related research in the future.