Sympatric populations of the genus Tupaia encompassing two cytotypes (cyt60, 2n = 60 and cyt62, 2n = 62) were found in the southern part of the Isthmus of Kra (the middle region of the Malay Peninsula, Thailand). C-bands, location of rDNA, and location of non-essential telomeric repeats (TRs) were investigated in detail for 23 animals captured in the area. Such chromosomal traits definitely reveal that two distinct cytotypes exist in the sympatric population, though the external morphological traits are similar. Hybrid cytotypes were not observed; thus, these two cytotypes appear to be genetically isolated sibling species. Chromosomal results compared with previous data, geographic distribution and morphological data observed with new insight suggest that, in the sympatric population, 'cyt60' represents members of Tupaia glis, while 'cyt62' identifies individuals of Tupaia belangeri. The cytogenetic information discovered in the present study offers new insight to morphological classification and, further, may provide substantial diagnostic characteristics for the distinction of tree shrew species.