This study investigates the gut microbiota of newly emerged adult females and males of five economically important Anastrepha species (Tephritidae) – A. ludens, A. obliqua, A. serpentina, A. striata, and A. fraterculus – by analyzing 16S rRNA amplicon sequences from 36 samples collected from ecologically relevant fruit hosts and locations in Mexico. We chose to concentrate only on newly emerged adults to identify bacteria that females could potentially transmit vertically to progeny via oviposition, a topic that remains poorly studied. Results revealed that Proteobacteria dominated the microbiota in all species, but substantial variation was observed in genus-level composition. Differentially abundant genera included Enterobacter, Gluconobacter, Tatumella, Providencia, Ochrobactrum, Siccibacter, Sphingobacterium, and Sphingobium. Significant differences in alpha diversity were observed between species, particularly between A. obliqua and A. striata, and between A. obliqua and A. serpentina based on the Shannon index. Anastrepha ludens, A. obliqua, and A. striata males exhibited higher species richness than females, although these differences were not statistically significant within individual species likely due to limited sample size. Interestingly, across all species, significant differences in microbiota composition were observed between males and females. Our findings suggest that morphological, physiological (i.e., metamorphosis) and ecological factors, such as possible gut structural differences and host fruit preferences, may influence the composition of the gut microbiota, potentially affecting the ecological adaptability and pest behavior of these flies.