Karyotype analysis was carried out on gill cells of three species of octopods using a conventional air-drying method. The karyotype results showed that all the three species have the same diploid chromosome number, 2n=60, but with different karyograms as 2n=38M+6SM+8ST+8T, FN (fundamental number)=104 (Cistopus
chinensis Zheng et al., 2012), 2n=42M+6SM+4ST+8T, FN=108 (Octopus
minor (Sasaki, 1920)) and 2n=32M+16SM+12T, FN=108 (Amphioctopus
fangsiao (d’Orbigny, 1839–1841)). These findings were combined with data from earlier studies to infer the genetic relationships between nine species via cluster analysis using the karyotype evolutionary distance (De) and resemblance-near coefficient (λ). The resulting tree revealed a clear distinction between different families and orders which was substantially consistent with molecular phylogenies. The smallest intraspecific evolutionary distance (De=0.2013, 0.2399) and largest resemblance-near coefficient (λ=0.8184, 0.7871) appeared between O.
minor and C.
chinensis, and Sepia
esculenta Hoyle, 1885 and S.
lycidas Gray, 1849, respectively, indicating that these species have the closest relationship. The largest evolutionary gap appeared between species with complicated karyotypes and species with simple karyotypes. Cluster analysis of De and λ provides information to supplement traditional taxonomy and molecular systematics, and it would serve as an important auxiliary for routine phylogenetic study.