We evaluated fidelity of various reverse transcriptases (RTs) by a novel method with modified next-generation sequencing (NGS). In the optimized condition, one NGS run could handle cDNA products from multiple cDNA synthesis reactions performed at different conditions. This was achieved using a primer containing not only the tag of 14 randomized bases to label each cDNA molecule but also a tag of five bases to label each reaction condition. With this method, we quantitated the error rates of 44 cDNA synthesis reactions by retroviral RTs or genetically engineered DNA polymerases with RT activity under different conditions. The results indicated that high concentrations of MgCl2, Mn(OCOCH3)2, and dNTP decrease the fidelity and that these effects are more pronounced in reactions using RT from human immunodeficiency virus type 1. This is the first report about a precise fidelity monitoring of various RTs by a direct sequence determination.