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Abstract Background The gut microbiota is known to play a role in a mosquito vector’s life history, a subject of increasing research. Laboratory experiments are essential for such studies and require laboratory colonies. In this study, the conservation of field-obtained midgut microbiota was evaluated in laboratory-reared Anopheles gambiae (s.l.) mosquitoes continuously hatched in water from field breeding habitats. Methods Pupae and late instars were obtained from the field and reared, and the emerged adults were blood-fed. The eggs obtained from them were hatched in either water from the field or in dechlorinated tap water. The mosquito colonies were maintained for 10 generations. Midguts of female adults from unfed F0 (emerging from field-caught pupae and larvae), F5 and F10 were dissected out and genomic DNA was extracted for 16S metagenomic sequencing. The sequences were compared to investigate the diversity and bacterial compositional differences using ANCOM and correlation clustering methods. Results Less than 10% of the bacterial families identified had differential relative abundances between generational groups and accounted for 46% of the variation observed. Although diversity reduced in F10 mosquitoes during laboratory colonization (Shannon-Weaver; P-value < 0.05), 50% of bacterial genera were conserved in those bred continuously in field-water compared to 38% in those bred in dechlorinated tap water. Conclusions To our knowledge, this study is the first report on the assessment of gut bacterial community of mosquitoes during laboratory colonization and recommends the use of water from the natural breeding habitats if they are intended for microbiota research.