Â© 2019 The Authors. Methods in Ecology and Evolution Â© 2019 British Ecological Society Tracking mosquitoes using current methods of markâ€“releaseâ€“recapture are limited to small spatial and temporal scales exposing major gaps in understanding long-range movements and extended survival. Novel approaches to track mosquitoes may yield fresh insights into their biology which improves intervention activities to reduce disease transmission. Stable isotope enrichment of natural mosquito breeding sites allows large-scale marking of wild mosquitoes absent human handling. Mosquito larvae that develop in 2H-enriched water are expected to be detectable for over 4 months using tissue mass fraction 2H measurements, providing opportunities for long-term markâ€“capture studies on a large scale. A laboratory study followed by a field experiment of mosquito larval habitat 2H enrichment was conducted in Mali, to evaluate potential labelling of wild mosquitoes. Twelve natural larval sites were enriched using [2H] deuterium oxide (D2O, 99%). Enrichment level was maintained by supplementation following dilution by rains. Availability of 2H to mosquito larvae was enhanced by locally collected and cultured microorganisms (i.e. protozoa, algae and bacteria) reared in deuterated water, and provided as larval diet. Putative natural predators were removed from the larval sites and first instar larvae Anopheles gambiae s.l. larvae were added every other day. Emergence traps enabled collection of eclosing adults. Adult mosquitoes were kept at laboratory conditions for analysis of label attrition with age. Deuterium enrichment of wild mosquitoes above background levels (maximumÂ =Â 143.1 ppm) became apparent 5â€“6Â days after initial exposure, after which 2H values increased steadily until c. 24Â days later (to a mean of c. 220Â ppm). Anopheles and Culex mosquitoes showed significantly different 2H values (211 and 194.2Â ppm, respectively). Both genera exhibited exponential label attrition (e(-x)) amounting to 21.6% by day 30 post-emergence, after which attrition rate continuously decreased. Males of both taxa exhibited a higher mean 2H value compared to females. Deuterium oxide proved useful in marking mosquitoes in their natural larval sites and although costly, may prove valuable for studies of mosquitoes and other aquatic insects. Based on our field study, we provide a protocol for marking mosquito larval sites using deuterium oxide.