Rapid determination of the larval species composition and understanding of their genetic structure is important to establish the appropriate management system for multiple species infesting in fruits. We established accurate and rapid diagnostic methods based on multiplex polymerase chain reaction (PCR) diagnostic techniques to discriminate the three major lepidopteran species in orchard, Carposina sasakii, Grapholita dimorpha, and Grapholita molesta. Each species was identified by amplifying species-specific PCR products (375 bp for C. sasakii, 125 and 234 bp for G. dimorpha, and 125 bp for G. molesta). Based on species composition analysis from six types of infested fruits, G. dimorpha constituted the highest proportion (47.8%), followed by 35.2 and 13.5% for G. molesta and C. sasakii, respectively. Interestingly, high prevalence was found in G. dimorpha and G. molesta for plum and peach, respectively. Based on genetic diversity analysis, the three insect species exhibited moderate or high haplotype diversity and low nucleotide diversity, ranging from 0.319 to 0.699 and 0.0006 to 0.0045, respectively. Demographic expansion was not detected according to either a neutrality test or mismatch distribution analysis. Moreover, no significant genetic structure corresponding to province, host plant, fruit type, or collection period was observed. These results suggest that the population of each species would have high dispersal ability following fruit-generating periods via intrinsic host adaptation ability regardless of the spatial and temporal conditions. Determination of larval composition on fruit is valuable for establishing appropriate management systems that take the species into consideration; additionally, population genetic approaches can be utilized to understand the effects of environmental factors (province, host fruit, fruit type, etc.) on population structures.