Species- and sex-related differences of four laboratory tephritid fruit fly (Diptera: Tephritidae) gut microbiomes evaluated with full-length 16S rRNA PacBio Kinnex sequencing. Insect gut microbiomes are shaped by multiple endogenous and environmental factors. Our study evaluated the impacts of how host sex and species influence the microbiome in laboratory-reared tephritids fruit flies when controlled for location, time, and adult diet. We evaluated the gut microbiome of four lines of pest tephritid fruit fly adults (Bactrocera dorsalis, Bactrocera latifrons, Ceratitis capitata, Zeugodacus cucurbitae) using near full-length 16S rRNA sequencing with a PacBio Kinnex concatenation-based approach. We analyzed groups of males and females from each species at the same set of time, across four timepoints in a core insectary. Results demonstrate a clear impact of fruit fly species on the gut microbiome composition of the different fruit flies. Furthermore, for B. dorsalis, B. latifrons, and C. capitata, we saw an influence of sex on amplicon sequence variant (ASV) composition. However, while there was a separation of samples between the sexes for each timepoint, there was no characteristic male or female microbiome in all cases.Dataset includes ASV count, taxonomy, fasta files, metadata, and R scripts. Raw sequence data has been deposited to the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) under the accession number PRJNA1196954.