The genetic diversity and species-diagnostic markers in the introduced apple snail, Pomacea canaliculata and in the native Thai apple snails; Pila ampullacea, P angelica, P pesmei, and P polita, were investigated by restriction analysis of COI and are reported for the first time. Twenty-one composite haplotypes showing non-overlapping distributions among species were found. Genetic heterogeneity analysis indicated significant differences between species (P < 0.0001) and within P pesmei (P < 0.0001) and P angelica (P < 0.0004). No such heterogeneity was observed in Pomacea canaliculata (P > 0.0036 as modified by the Bonferroni procedure), P ampullacea (P = 0.0824-1.000) and P polita (P = 1.0000). A neighbor-joining tree based on genetic distance between pairs of composite haplotypes differentiated all species and indicated that P angelica and P pesmei are closely related phylogenetically. In addition, the 16S rDNA of these species was cloned and sequenced. A species-specific PCR for P canaliculata was successfully developed with a sensitivity of detection of approximately 50 pg of the target DNA template. The amplification of genomic DNA (50 pg and 25 ng) isolated from the fertilized eggs, and juveniles (1, 7, and 15 d after hatching) of Pomacea canaliculata was also successful, and suggested that Pomacea canaliculata and Pila species can be discriminated from the early stages of development.