Somatic gene V617F mutation in JAK2 is a critical molecular and biological indicator to diagnosis of chronic myeloproliferative disease (MPD). This study was aimed to investigate the genetic background of V617F mutation in 46/1 gene haplotype in Chinese MPD patients, and the frequencies of 46/1 gene haplotype and V617F mutation in three nationalities of Chinese populations. Peripheral blood or bone marrow samples of 150 V617F mutation positive MPD patients, 123 V617F mutation negative MPD patients, 124 healthy Han individuals, 395 healthy Tibetan individuals and 315 healthy Yugu individuals were collected. The allele-specific multiplex PCR method was established, the presence or absence of V617F mutation, the presence or absence of 46/1 haplotype, and the relationship between V617F and 46/1 haplotype were easily identified by agarose gel image. The results showed that the V617F mutation located in the 46/1 haplotype of 88 cases (58.67) among 150 V617F-positive MPD cases. In 814 Chinese healthy individuals including Han, Tibetan, Yugu nationalities, the frequency of the 46/1 gene haplotype was 38.37 without difference in the frequency among different nationalities, and no V617F mutation was found in Chinese healthy populations, The frequency of the 46/1 gene haplotype was 43.09 in V617F mutation negative MPD patients and was 69.33 in V617F mutation positive MPD patients, the latter was obviously higher than former and than that in healthy Han individuals. In conclusion, a multiplex PCR method has been developed that is simple and useful to identify V617F mutation in JAK2 gene and its relationship to the 46/1 haplotype. In more than half of Chinese V617F-positive MPD patients, the V617F mutation locates in 46/1 haplotype in JAK2. The frequencies of 46/1 haplotype are statistically insignificant among Han, Tibetan and Yugu nationality populations.