Ambiguity of morphological characteristics made distinguishing the two subspecies of Liniparhomaloptera disparis(Cypriniformes: Homalopteridae) difficult. Further morphological evidence in infralabial shape and head spot pattern of the loach were herein provided for the first time to define the subspecies, based on laboratory observation of 441 specimens from 15 drainage systems, including the three western, northeastern, and eastern major rivers on Hainan Island. We recognized four head patterns of dark spots: small spots, vermiform spots, mixture of small and vermiform spots, and large spots. The small-spotted head pattern occurred in all seven populations east of the Yunkai Mountains, as well as in the only eastern population on Hainan Island. The vermiform-spotted head pattern occurred in all five populations west of the Yunkai Mountains. The mixed head pattern of small and vermiform spots occurred in both the western and northeastern populations on Hainan Island, but the large-spotted head pattern was also found in the western population. Additionally, we recognized three infralabial shapes: semicircular, subtrapezoidal, and horseshoe-like. The semicircular infralabium occurred in all five populations west of the Yunkai Mountains. The subtrapezoidal infralabium occurred in all seven populations east of the Yunkai Mountains. The horseshoe-like infralabium occurred in all three populations on Hainan Island. Therefore, populations east of the Yunkai Mountains had distinctly small-spotted head pattern and subtrapezoidal infralabia in combination, defined as the nominate subspecies L. disparis disparis according to the holotypic locality. Surprisingly; populations on Hainan Island were distinctly differentiated from the nominate mainlanders; all had uniquely the horseshoe-like infralabium, but the eastern population had small-spotted head pattern like the nominate mainlanders, the northeastern population had the mixed-spotted head pattern solely, and the western population had both the mixed-spotted and large-spotted head patterns. Based on the subspecific type locality(Wanquan River), we have redefined all three populations on Hainan Island that had mostly the mized-spotted head pattern and horseshoe-like infralabium in combination as the second subspecies L. disparis qiongzhongensis. Populations west of the Yunkai Mountains had distinctly the vermiform-spotted head pattern and semicircular infralabium in combination. This morphological variation may have resulted from the orogeny of the Yunkai Mountains. The differences in head spot pattern among populations on Hainan Island and the differences in infralabial shape either between mainland and Hainan Island or between east and west of the Yunkai Mountains suggest geographic isolation of rivers may have occurred long ago but at different times. This paper demonstrates the infralabial shape as a taxonomic character for the first time; this character in combination with the previous characters like head spot pattern can be used to identify subspecies or geographic populations effectively. Molecular analyses may prove the mainland populations west of the Yunkai Mountains and the populations on Hainan Island are genetically unique, new subspecies, and their geophylogenetic associations.