For the majority of Rhinolophus species a karyotype consisting of either 58 or 62 chromosomes was reported. The members of the R. trifoliatus clade are therefore distinguished from other rhinolophids by low diploid chromosome numbers (2n) and a high rate of chromosomal changes. Recently, based on cytogenetic characters, a cryptic species, R. luctoides, was described and a subspecies, R. luctusmorio, was elevated to species rank. In the present work, the karyotypes of a male bat from Vietnam and a female one from China, both classified as R. luctus by morphological characters, were studied by G-banding and fluorescence in situ hybridization with whole chromosome painting probes derived from Aselliscus stoliczkanus. In the male specimen from Vietnam, the composition of the 15 autosomal bi-armed pairs (2n = 32, FN = 60) is identical to that of R. luctoides from Malaysia. For reason of differences in body size and baculum length as well as in skull morphology, this specimen is provisionally treated as R. cf. luctoides. The karyotype of the female specimen from Sichuan province, China, differs from R. luctoides by a whole arm reciprocal translocation resulting in an altered arm composition of pairs 3 and 4. For this reason, the elevation of the Chinese subspecies of R. luctus, i.e. R. lanosus, to species rank is suggested. The karyotype of the endemic Taiwanese species R. formosae with a diploid number of 2n = 52 is mainly composed of acrocentric autosomal pairs. Of the five bi-armed pairs, only the two smallest show the same arm composition as found in the 2n = 32 karyotypes of other members of the R. trifoliatus clade. The composition of the remaining three bi-armed pairs is unique and represents an apomorphic feature for R. formosae.