There has been considerable interest in understanding biological, ecological, historical, and evolutionary processes that contribute to the diversification of species and populations among tephritid fruit flies. Only a limited number of studies have examined the genetic diversity and population biology of species belonging to the genus Anastrepha considering fine-scale differentiations associated to locality as well as hosts over an entire fruiting season.To expand our understanding of population structure and genetic diversity in one of the critical Anastrepha fruit flies populations in a highly diverse tropical environment we analyzed Anastrepha obliqua (Macquart) (Diptera: Tephritidae) in the Mexican state of Veracruz from five host fruit species and 52 geographic collections using sequence data from mtDNA and microsatellite markers from nuclear DNA. Indeed, we examined the population structure of this pest in a micro-geographic region and report on relationships and historical processes for individuals collected within a small portion of the geographic range of its distribution. Analyses of 1055 bp mtDNA sequences from CO1and ND1genes across 400 individuals detected 34 haplotypes. Haplotype and nucleotide diversity was low, with 53% of the individuals exhibiting a single haplotype (OBV1). Host association and fine-scale differentiation at 17 microsatellite markers across 719 individuals from 32 of the 52 geographic collections reveal fragmented A. obliqua populations. These findings have important implications for the implementation of the Sterile Insect Technique (SIT) and other pest management programs used to control this pestiferous fruit fly.