Biological invasion by non-native subspecies or populations is one of the most serious threats to ecosystems, because these species might be easily established in the introduced area and can negatively affect native populations through competition and hybridization. Pale chub Opsariichthys platypus, one of the most common fish in East Asia, exhibits clear genetic differentiation among regional populations; however, introgression and subsequent loss of genetic integrity have been occurring throughout Japan due to the artificial introduction of non-native conspecifics. In this study, we developed a simple SNP genotyping method to discriminate between native and non-native mitochondrial DNA (mtDNA) haplotypes in pale chub using real-time PCR assay. We then investigated the distribution patterns of non-native pale chub in Tokai region, located in the center of Honshu Island, Japan and developed a predictive model of the occurrence of non-natives to reveal the factors influencing their invasion. The specificity and accuracy of the genotyping method were confirmed by using samples whose haplotypes were determined previously. Extensive occurrence of non-native haplotypes in Tokai region was detected by this method. In addition, our models suggested that the presence of non-natives varied greatly depending on the river system, and was positively influenced by the impounded water areas. Our method could accurately distinguish between native and non-native haplotypes of pale chub in Japan and suggested key environmental factors associated with the presence of non-natives. This approach can greatly reduce experimental costs be a great contribution for quantitative investigation.