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Endosymbiotic Wolbachia bacteria are known to influence the host physiology, microbiota composition, and dissemination of pathogens. We surveyed a population of Tabanus nigrovittatus, commonly referred to as "greenheads," from Crane Beach (Ipswich, MA, USA) for the presence of the alphaproteobacterial symbiont Wolbachia. We studied the COI (mitochondrial cytochrome oxidase) marker gene to evaluate the phylogenetic diversity of the studied specimens. The DNA sequences show strong similarity (between 99.9 and 98%) among the collected specimens but lower similarity to closely related entries in the NCBI database (only between 96.3 and 94.7%), suggesting a more distant relatedness. Low levels of Wolbachia presence necessitated a nested PCR approach, and using 5 markers ( ftsZ , fbpA , dnaA , coxA , and gatB ), we determined that two recognized "supergroups" of Wolbachia species were represented in the studied specimens, members of clades A and B. Using next-generation sequencing, we also surveyed the insect gut microbiomes of a subset of flies, using Illumina and PacBio 16S rRNA gene sequencing with barcoded primers. The composition of Proteobacteria also varied from fly to fly, with components belonging to Gammaproteobacteria making up the largest percentage of organisms (30 to 70%) among the microbiome samples. Most of the samples showed the presence of Spiroplasma , a member of the phylum Mollicutes , although the frequency of its presence was variable, ranging from 2 to 57%. Another noteworthy bacterial phylum consistently identified was Firmicutes , though the read abundances were typically below 10%. Of interest is an association between Wolbachia presence and higher Alphaproteobacteria representation in the microbiomes, suggesting that the presence of Wolbachia affects the host microbiome. IMPORTANCE Tabanus nigrovittatus greenhead populations contain two supergroups of Wolbachia endosymbionts, members of supergroups A and B. Analysis of the greenhead microbiome using next-generation sequencing revealed that the majority of bacterial species detected belonged to Gammaproteobacteria , with most of the samples also showing the presence of Spiroplasma , a member of the Mollicutes phylum also known to infect insects. An association between Wolbachia presence and higher Alphaproteobacteria representation in the microbiomes suggests that Wolbachia presence affects the host microbiome composition.