Abstract Earlier we have reported two G6PD variants viz.; G6PD Jamnagar and G6PD Rohini. The enzymes from both the variants showed altered biochemical properties with mild enzyme deficiency and were classified as unique Class III variants. G6PD Jamnagar was found to be associated with drug-induced hemolytic anemia whereas G6PD Rohini was picked up during a population survey. Subsequent molecular studies on the DNA from both the cases showed the presence of the Kerala–Kalyan (949 G→A) mutation. Hence, this study besides supporting the fact that biochemically distinct variants could have the same mutation at the molecular level also highlights the importance of molecular characterization of G6PD variants.