With increasing urbanization, organisms face a myriad of novel ecological challenges. While the eco-evolutionary dynamics of urbanization are currently receiving a great deal of attention, the effect of urban disturbance on the microbiome of urban organisms is relatively unstudied. Indeed, studies of the microbiome may illuminate the mechanisms by which some species thrive after urbanization (pest implications), while other species go locally extinct (biodiversity implications). We investigated the gut microbiome of the Western black widow spider (Latrodectus hesperus). L. hesperus is an ideal model system as they are a pest species of medical importance in urban ecosystems, often forming dense urban infestations relative to the sparse populations found in their native Sonoran Desert. To gain insight into the composition of the microbiome in L. hesperus and its potential function, we sampled 4 urban, 4 desert, and 2 laboratory-reared spiders, and high-throughput sequencing of the 16S rRNA V4 region was used to investigate the diversity of gut microbiota. Dominant bacterial phyla across all samples were Firmicutes, Proteobacteria, and Actinobacteria. While desert widows showed more gut microbial diversity than urban widows, the difference was not statistically significant. The relative abundance of taxonomic classes Blastocatellia, Acidobacteriia, and Thermoleophilia detected in desert spiders was especially higher than those in urban and laboratory-reared spiders. However, urban spiders had a higher relative abundance of taxonomic class Actinomycetia. Differences in widow gut microbiome diversity improves our understanding of how features unique to a habitat, like prey diversity and soil microbes, may be shaping their microbiome. Additionally, this work further highlights the impact urbanization has on biodiversity loss, which indirectly develops a new biomarker for differentiating between urban and desert black widow spiders based on their gut microbiome.