5,10-methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in folate metabolism. A novel polymorphic site in MTHFR (G1793A) could influence the homocysteine levels and was first described in 2002. Investigations revealed that this allele was associated with susceptibility to several cancers, but its distribution around the world was not adequate. To study the prevalence of the mutant frequency in Chinese populations, 923 healthy individuals from 13 Chinese populations distributing widely from north to south were collected. DNA samples were isolated from peripheral blood samples and genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), with the digestion of restriction endonuclease BsrBI. Of the 923 individuals, 82.1% were GG homozygous, 17.2% were GA heterozygous and 0.7% were AA homozygous. The frequency of the MTHFR 1793A allele in all tested individuals was 9.3%, which was slightly lower than indicated by HapMap (10%, Beiing Han, 45 samples). The frequencies of A allele were generally higher in southern China than that in northern China, and the frequencies had significant variance in 13 Chinese populations (X2 = 26.315, P = 0.010). Summarizing of the MTHFR G1793A allele polymorphism, including control groups in the case-control studies, we found only 20 normal peoples with AA homozygous (7 Chineses, 1 Caucasian, 2 Java Indonesias, 2 non-Hispanic whites, 6 Irish women, 2 Indians). The Java Indonesias and Ashkenzai Jevish had the highest (26.6%) and the lowest (1.3%) 1793A frequency, respectively. Together with our previous data, the MTHFR G1793A polymorphism was in linkage disequilibrium with both C677T and A1298C polymorphism sites in Chinese population, but not as strong as presented by HapMap.