[Objectives] Intestinal flora of animals is related to their health, and the composition of flora is affected by many factors including living environment. Exploring the relationship between intestinal flora diversity and living condition is conducive to promoting animal conservation. Based on non-injury sampling and 16S rDNA high-throughput sequencing, this study compared the intestinal flora structures of captive Canis lupus at two different geographical latitudes. [Methods] High-throughput sequencing by the Illumina Miseq platform was performed on 19 fecal samples of C. lupus collected from Tianjin Zoo (38°88′ N) and Nanjing Hongshan Forest Zoo (31°14′ N) (Tianjin group and Nanjing group, respectively). Bioinformatics was used to analyze the sequencing and to compare the differences in microbial composition and diversity between two groups. Student’s t test and Welch’s t test were used to detect the difference in the Alpha diversity, while the Beta diversity was analyzed by principal coordinates analysis (PCoA) based on weighted and unweighted UniFrac distances. Functional prediction analysis was used to predict the metabolic pathways and explore the correlation with the intestinal microbiota of C. lupus of both groups. [Results] After OTU clustering according to 97% similarity (Fig. 1), the microbial composition was analyzed between two groups. At the phyla level, Firmicutes and Actinobacteria were the dominant phyla in two groups, and the abundance of Campilobacterota in Tianjin group was higher than that in Nanjing group (Fig. 2). At the genus level, the relative abundance of Peptostreptococcus in Nanjing group was significantly higher than that in Tianjin group (Fig. 3). Peptoclostridium, Collinsella and Clostridium _sensu_stricto_1 were the dominant genus for both groups (Fig. 4). Both the Alpha diversity indices (Shannon index) (Fig. 5, 6) and PCoA analysis based on unweighted UniFrac distance revealed significant group differences in intestinal flora structure (Fig. 7). The main functions of intestinal microbiota of two groups were basically the same (Fig. 8). [Conclusion] The intestinal flora diversity of Nanjing group was significantly higher than that of Tianjin group. Different feeding environment directly affects the diversity and relative abundance of intestinal flora. The findings in this study provide basic scientific reference for both the conservation of captive population and the comprehensive management of wild population in this species.