Two North American species of mosquitofish, the Western (Gambusia affinis Baird and Girard, 1853) and Eastern mosquitofish (G. holbrooki Girard, 1859), rank amongst the most invasive freshwater fishes worldwide. While the existing literature suggests that G. affinis was introduced to mainland China, empirical evidence supporting this assumption was limited, and the possibility remained that both species were introduced during campaigns attempting to reduce vectors of malaria and dengue fever. We used combined molecular information (based on phylogenetic analyses of sequence variation of the mitochondrial cytochrome b gene) and morphometric data (dorsal and anal fin ray counts) to confirm the presence of only one species, G. affinis, across ten sampling sites in mainland China.