Objective To analyze the polymorphism of G6PD (glucose-6-phosphate dehydrogenase) gene mutation in coding regions and its correlations with the proneness to primaquine hemolysis in vivax malaria patients in Yunnan Province, China.Methods Blood samples were collected from the vivax malaria patients receiving an treatment regime of “eight-day therapy of chloroquine/primaquine” in Yunnan Province in 2018. Patients with “tea-colored” urine and reduced G6PD enzyme activity during the primaquine therapy were defined as primaquine-induced acute hemolysis. Human genomic DNA was extracted from the blood samples, and of the G6PD gene fragments containing 12 exon were amplified by PCR and sequenced. The sequences were compared with the nonmutation-type sequence, edited and spliced by DNAStar 11.0 and BioEdit 7.2.5 softwares to generate the cDNA sequence of G6PD gene. MEGA 5.04 and DnaSP 5.10 softwares were used to analyze the mutation polymorphism the selection effect of the cDNAs. The correlation coefficient (r) and odds ratio (OR) for primaquine-induced hemolysis was estimated.Results A total of 184 blood samples were collected, of them 44 generated complete G6PD cDNA strand sequence (length, 1 545 bp), including one sample from a hemolysis case. Alignment of the cDNA chain with the nonmutation-type sequence revealed the mutation sites including c.461 T > A, c.574 C > T, c.786 C > T, c.1059 C > T, c.1311 T > C, and c.1376 G > T, with a frequency of 1.5/100 000, 1.5/100 000, 1.5/100 000, 1.5/100 000, 45.6/100 000 and 1.5/100 000, respectively. The two-site mutation linkage between c.1311 and c.1376 was only present in the genome of the hemolysis cases, and the c.1376 site mutation was positively correlated with the primaquine-induced hemolysis (r = 1.000, P > 0.05). The 44 cDNA strands were defined into 6 haplotypes (Hap_1-Hap_6), with an expected heterozygosity, nucleic acid diversity index π, and Ka/Ks ratio were 0.493, 0.001, and 0.062, respectively. The Hap_2 type containing c.1311 single-site mutation accounted for the highest proportion of (68.2%, 30/44). The site c.1311 with high frequency of mutation had the most complex allele composition, comprising 5 types of zygote including nonmutation hemizygote (9/44, 20.4%), mutant hemizygote (16/44, 36.4%), nonmutation homozygote (5/44, 11.5%), mutant heterozygote (10/44, 22.7%), and mutant homozygote (4/44, 9.1%). The OR value of c.1311 mutation to the primaquine-induced hemolysis was 0.6879 (P > 0.05), showing no correlation in between. The D value of Tjima’s neutral test was -1.414 (P > 0.05), and the D value of the whole fragment was < 1, indicating that the mutations detected in the sequence were not a non-neutral mutation under directional selection pressure; meanwhile the Ka/Ks ratio was 0.062, indicating that the sequence was highly conservative and the frequency of missense mutation was lower than that of synonymous mutation.Conclusion In malaria patients in Yunnan Province, the T > C base substitution at c.1311 is a common mutation in G6PD coding region, but only the G > T mutation at c.1376 is positively correlated with the occurrence of oxidant induced hemolysis.