Natural occurrence of pentaploid loach, Misgurnus anguillicaudatus discovered from Liangzi Lake area, Hubei Province, central China, is reported here for the first time. The evidences from karyotyping, DNA content analysis and nuclear volume measurements were described to confirm the pentaploid nature of the identified individual. This individual was phenotypically indistinguishable from its sympatric diploid and tetraploid counterparts. The chromosome number was 5n = 125, the mean erythrocyte nuclear DNA content was 2.62 and 1.25 times of their diploid and tetraploid counterparts, respectively. An origin of such polyploidy form either from genome addition of normal sperm nuclei to unreduced egg and the mating of tetraploid individual and hexaploid individual, respectively, is hypothesized.