Highlights Rainwater harvesting systems can harbor mosquitoes and their larvae. Approximately half of sampled systems were found to contain mosquito larvae. Mosquitoes were more abundant in systems with unscreened openings and multiple inlet filters. Frequent maintenance and eliminating unscreened openings minimize mosquito presence. Abstract. Mosquitoes are associated with the spread of diseases such as the Zika and West Nile viruses. Government and health officials recommend the elimination of standing water to prevent the breeding of mosquitoes that transmit these viruses. Previous research has shown that rainwater harvesting (RWH) systems in high-rainfall regions tend to be underutilized, thereby creating a source of standing water that could potentially harbor mosquitoes and their larvae. As RWH is an important tool for conserving potable water and mitigating stormwater runoff, it is imperative to determine if these systems are contributing to the proliferation of mosquitoes and, if they are, to identify measures to prevent this. The prevalence and relative abundance of mosquitoes were investigated in 64 RWH cisterns throughout Virginia and North Carolina. Mosquito larvae captured during sampling were identified morphologically to genus and species. Of the 64 systems sampled, 47% contained mosquitoes; the predominant species found was Aedes albopictus, a potential carrier of Eastern Equine Encephalitis, LaCrosse Encephalitis, West Nile virus, and Zika virus. Systems with multiple inlet filters, unscreened storage tank openings, and/or heavy foliage in the immediate vicinity were most likely to contain mosquito larvae. Study results indicate that frequent maintenance, the elimination of unscreened tank openings, and the eradication of sources of standing water in the area surrounding a system greatly reduce the likelihood of mosquitoes proliferating in rainwater harvesting systems. Keywords: Container breeding, Mosquito, Mosquito larvae, Rainwater harvesting.