This study was carried out to shed light on confused subfamilial groupings in the Cyprinidae from the biochemical viewpoint at the molecular level, specifically by using two-dimensional gel electrophoresis of liver proteins. Six pairs of cypriniform fishes, which are different from one another at familial, subfamilial, generic, specific, subspecific, and individual levels, were compared. The genetic distances between pairs of fishes increased as taxonomic ranks of the pairs became higher, confirming the reliable usefulness of this technique. Four species representing the different subfamilies, Cyprininae, Gobioninae, Acheilognathinae, and Leuciscinae, were compared to give new insight into relationships at the subfamilial level. Cyprinus carpio (Cyprininae) and Pseudogobio esocinus esocinus (Gobioninae) gave the smallest genetic distance and the largest values were obtained between either one of the above species and Acheilognathus melanogaster (Acheilognathinae), suggesting that the former two subfamilies compose the most closely related group that is in turn distantly related to Acheilognathinae. Tribolodon hakonensis (Leuciscinae) had almost equal genetic distances to the three other species.