In order to study Leopard Cat (Prionailurus bengalensis) genomics and protect endangered Leopard Cat, the biological characteristics including the morphology, the adherence rate, the frozen survival rate, the growth curve and the cell karyotype of in vitro cultured fibroblast cells were investigated. Three types of Leopard Cat tissues of cartilage, heart and lung were collected. The adhesive culture with tissue block was carried out for the primary culture of somatic cells, and then subculture was conducted after digestion by the trypsin. The frozen cell viability was calculated by cell counting method and the growth curve was made. The chromosome karyotype and G band were analyzed by normal chromosome specimen preparation technology. Fibrod cells were observed from D3 of cartilage tissue block primary culture and they overspread all the bottom surface of cultured flask by Day 6﹣7, while these events took place by D5 and D12 of heart tissue culture, and by D4 and D8﹣9 of lung tissue culture (Fig. 1). The cells derived from three organs or tissues all exhibited characteristics of fibroblast cells (Fig. 2, 3). The adherence speed following culture showed lowest to fastest from the heart-derived cells, lung-derived cells and cartilage-derived cells, respectively (Fig. 4). Comparing the frozen survival rates of three different sources of somatic cells, we showed significant reduction from P6 to P12 in both cartilage-derived cells (P6﹣P12: 91.0%﹣97.6% vs. 76.8%﹣85.5%, P < 0.05) and the heart-derived cells (P6﹣P12: 82.7%﹣88.1% vs. 43.7%﹣80.5%, P < 0.05). Lung-derived cells also showed similar trend but no significant difference was observed from P6 to P12 (P6﹣P12: 83.4%﹣96.8% vs. 73.9%﹣93.3%, P > 0.05) (Table 2). The growth curve analysis of three types of fibroblast cells showed an “S-model”, the cartilage-derived cells proliferated faster, next by the lung-derived cells, and the cardiac-derived cells proliferated slower (Fig. 5). The results of karyotype analysis showed that the number of chromosomes of all three different sources of fibroblast cells were 2n = 38 (Fig. 6), including 18 pairs of autosomes with 6m + 10sm + 2st types, one pair of sex chromosomes, and X chromosome was m type (Fig. 7, Table 3). This study established three fibroblast cell lines derived from three tissues of leopard cat, and analyzed their biological characteristic and karyotype. The results provide important biological information for future genetic research and species protection of leopard cat.