Approximately 18 million people live in malaria-endemic areas with 218,450 reported confirmed cases and 161 reported deaths in Indonesia. Currently, primaquine (PQ), the 8-aminoquinolines, is still the only drug for radical cure and preventing relapse of malaria. However, the individuals with G6PD deficiency (G6PD-d) have risk of hemolysis. Currently, few data of the prevalence of G6PD-d and genotyping are available. This study will provide the prevalence of G6PD-d and the genotyping in malaria cases in South Central Timor (TTS) district. G6PD status was analyzed with quantitative (Randox G6PD test, UK) follow with PCR-RFLP and sequencing to identify the variant of G6PD-d genotyping. Malaria was confirmed by n-PCR (Promega, Madison, USA). A total 64 of 181 individuals with G6PD-d from South Central Timor (TTS) district were analyzed. About 25 of 64 cases of G6PD-d were tested positive for malaria with P. vivax as the dominant species 56% (14/25) and most of the cases were female 73.3% (11/15). Among the 64 G6PD-d the genotyping Vanua Lava (10,883 T>C) WHO classifies G6PD-d genetic variants class II with severe deficiency <10% the enzyme activity were dominant. The variant of Vanua Lava is dominant and the high G6PD-d indicated that screening for G6PD deficiency is necessary.