The Japanese fauna of the yellow butterfly Eurema hecabe (L.) (Lepidoptera; Pieridae) populations in Ryukyu area (Southwestern Islands) were shown to differ from their Mainlands population (Honshu) in its seasonal forms of adult wing color pattern expression and in larval food habits. Recently, E. hecabe was divided into two species, E. hecabe and Eurema mandarina (de l'Orza) (Lepidoptera: Pieridae), based on their morphological, behavioral and genetic characteristics. However, whereas these two species are classified into two distinct groups based on nuclear DNA, both species are mixed in the phylogenetic tree from their mitochondrial DNA (mtDNA) sequences. The reason is a selective sweep by Wolbachia infection, and they can not be distinguished by DNA barcode method using mtDNA. We analyzed allozyme variation as a nuclear gene phenotype in 15 loci from 11 enzymes of the two closely related butterflies. Examining the esterase zymogram of each population, appearance frequency of Est-2 locus in each population varied greatly. E. mandarina showed the bands but E. hecabe showed no bands in Est-2 locus. The UPGMA and NJ phylogenetic trees branched out into two species—E. hecabe and E. mandarina—although their habitat overlapped in Okinawajima Is. populations. The results showed that the allozyme analyses were not affected by the selective sweep of Wolbachia infection between these two species.