Simple SummaryThe Mexican wolf (Canis lupus baileyi) is an endangered canine. Both Mexico and the United States are currently collaborating to reproduce and reintroduce individuals to their original habitats. However, keeping these wolves in captivity represents a great commitment to meet their basic needs. Diet is a determining factor that is closely related to health and reproductive fitness. The type of diet that is fed to canines in captivity must provide the required nutrients for their development and welfare. The study of the fecal microbiota is a non-invasive way to establish the abundance and diversity of bacterial communities to determine if they are in a healthy condition. We analyzed data from two captive populations of Mexican wolves (i.e., northern and central Mexico) receiving different type of diets (Michilia population: mainly kibble vs. Ocotal population: mainly raw meat). The operational taxonomic units (OTUs) in Michilia resulted in 204 genera and 316 species, while in Ocotal there were 232 genera and 379 species. In the Michilia, dominance of bacteria that degrade carbohydrates was observed (related to kibble diet). In contrast, the Ocotal microbiota was dominated by protein-degrading bacteria (related to raw meat diet). The main outcomes generated in this study should help to enhance the welfare of the captive Mexican wolves to increase its numbers.The Mexican wolf (Canis lupus baileyi) was once distributed in southern United States and northern Mexico. It is an endangered subspecies detached from the gray wolf, and likely exemplifies one of the original migration waves of C. lupus into the new world. This is a canine whose individuals survive in specialized facilities, zoos, and museums as part of captive-breeding programs. In order to contribute to the improvement of the management of this species and favor its long-term conservation in Mexico, we aimed to evaluate the diversity and abundance of the fecal bacterial microbiota in two populations exposed to different types of diet: (1) Michilia (23° N, 104° W); kibble daily and raw meat sporadically, and (2) Ocotal (19° N, 99° W); raw meat daily and live animals periodically. Next generation sequencing (V3-V4 16S rRNA gene) by Illumina was implemented. The operational taxonomic units (OTUs) in Michilia resulted in 9 phyla, 19 classes, 34 orders, 61 families, 204 genera, and 316 species, while in Ocotal there were 12 phyla, 24 classes, 37 orders, 69 families, 232 genera, and 379 species. Higher estimated Chao1 richness, Shannon diversity, and core microbiota were observed in Ocotal. Differences (p < 0.05) between populations occurred according to the Bray–Curtis beta diversity index. In the Michilia, dominance of bacteria that degrade carbohydrates (Firmicutes, Lachnospiraceae, Blautia, Clostrodium, Eisenbergiella, Romboutsia, and Ruminococcus) was observed; they are abundant in kibble diets. In contrast, the Ocotal microbiota was dominated by protein-degrading bacteria (Fusobacteria, Fusobacteriaceae, and Fusobacteria), indicating a possible positive relation with a raw meat diet. The information generated in this study is fundamental to support the implementation of better management plans in the two populations considered here, as well as in different facilities of southern United States and Mexico, where this subspecies is kept in captivity for conservation purposes.