BackgroundHost-gut microbiota interactions are complex and can have a profound impact on the ecology and evolution of both counterparts. Several host traits such as systematics, diet and social behavior, and external factors such as prey availability and local environment are known to influence the composition and diversity of the gut microbiota.MethodsIn this study, we investigate the influence of systematics, sex, host size, and locality/habitat on gut microbiota diversity in five lizard species from two different sites in Portugal:Podarcis bocageiandPodarcis lusitanicus, living in syntopy in a rural area in northern Portugal (Moledo); the invasivePodarcis siculusand the nativePodarcis virescens, living in sympatry in an urbanized environment (Lisbon); and the invasiveTeira dugesiialso living in an urban area (Lisbon). We also infer the potential microbial transmission occurring between species living in sympatry and syntopy. To achieve these goals, we use a metabarcoding approach to characterize the bacterial communities from the cloaca of lizards, sequencing the V4 region of the 16S rRNA.ResultsHabitat/locality was an important factor explaining differences in gut bacterial composition and structure, with species from urbanized environments having higher bacterial diversity. Host systematics (i.e., species) influenced gut bacterial community structure only in lizards from the urbanized environment. We also detected a significant positive correlation between lizard size and gut bacterial alpha-diversity in the invasive speciesP. siculus, which could be due to its higher exploratory behavior. Moreover, estimates of bacterial transmission indicate thatP. siculusmay have acquired a high proportion of local microbiota after its introduction. These findings confirm that a diverse array of host and environmental factors can influence lizards’ gut microbiota.