As a continental island, Hainan Island is geographically isolated from the surrounding mainland. Exploring the genetic differentiation of insects among this island and the surrounding mainland will help us to further understand the effects of geographical isolation on genetic diversity. In this study, samples of two moth species (Lepidoptera: Sphingidae) were collected from 15 locations in the Hainan Island and the surrounding mainland, including 88 samples of Callambulyx rubricosa and 57 of Parum colligata. Two mitochondrial genes COI, Cytb and one nuclear gene EF-1α were selected as molecular makers to proceed following genetic diversity and differentiation analyses. The results based on the COI-Cytb analyses showed that the nucleotide diversity of the C. rubricosa and P. colligata was 0.00434 and 0.00818, respectively. There were obvious phylogeographic structures among the populations in Guangdong-Guangxi, Vietnam, and Hainan Island. Both species showed weak gene flow (N_m were 0.09-1.22 and 0.18-0.57, respectively) and distinct genetic differentiation (F_ST were 0.29-0.84 and 0.47-0.73, respectively) between these three populations. The highest genetic differentiation was detected between Hainan Island and the mainland populations. Meanwhile, the number of haplotypes in Hainan Island population was less than that of the surrounding mainland populations. Moreover, Mantel tests indicated that there were significantly positive correlations between genetic distance and geographic distance, and neutrality tests suggested that neither species has undergone significantly population expansion. In conclusion, our results implied that there were flaccid gene flows and distinct genetic differentiation of C. rubricosa and P. colligata between Hainan Island population and the surrounding mainland populations. The geographic isolation could be one of the important reasons for these noticeable differentiation, which might provide valuable advice for the biodiversity conservation in Hainan Island.