Ficus pumila var. pumila and Ficus pumila var. awkeotsang are two varieties of the same species (Moraceae); one is the original variant while the other is a later variant and, they have long been considered to interact with the same species of pollinator. However, the recent studies in morphology, ecology and pollination biology suggest that a differentiation between the pollinators of Ficus pumila var. pumila and Ficus pumila var. awkeotsang wasps may have occurred during the evolution. In the present studies, we used three molecular markers including 28SrDNA D1-D3, the mitochondrial Cytb and COI genes to investigate the genetic relationship of these two pollinators. The pollinator samples were collected from Ficus pumila var. pumila at three different locations as well as three cultivars of Ficus pumila var. awkeotsang. The data are summarized as follows: (1) The distribution of the four bases including A, T, G, C in the 28SrDNA sequence from the pollinators of Ficus pumila var. pumila and Ficus pumila var. awkeotsang is relatively akin. The average content of C + G (56%) is slightly higher than the average content of A+T (44%). The average content of A+T in the mitochondrial Cytb sequence is 76.1%, significantly higher than the average content of C+G (23.9%). The COI sequence analysis shows a similar result, with the A+T content (71.9%), higher than the G+C content (28.1%). This base composition pattern is consistent cross along hymenopteran mitochondrial sequences. Furthermore, in the Cytb and COI sequences, it appears to have the highest content of A/T base at the third position of a codon. (2) The 28SrDNA sequence evolution appears to be slower than the Cytb and COI gene sequences, is relatively conservative, and maybe more suitable for senior-order (family or subfamily) phylogenetic studies. In contrast, the Cytb and COI molecular markers may be more suitable for the genetic relationship analyses between the pollinators of Ficus pumila var. pumila and Ficus pumila var. awkeotsang due to their close ties. (3) The results derived from genetic distance analyses show that the average distance of the Cytb sequence of each sample within Ficus pumila L. pumila pollinator or within Ficus pumila var. awkeotsang pollinator is 0.0054 or 0.0164. The average genetic distance of the Cytb sequence between the two pollinator groups is 0.1385. The average genetic distance of the COI sequence of each sample within Ficus pumila var. pumila or Ficus pumila var. awkeotsang is 0.0048 or 0.0102, while the average genetic distance between the two wasp populations is 0.1896. Since the genetic distance between the two wasp groups are significantly greater than the genetic distance within the respective group, these data suggest that the two groups of fig wasps have differentiated during the evolution and reached to the level of species identification, namely the pollinators of Ficus pumila var. pumila and Ficus pumila var. awkeotsang are the two different wasp species.