This study employed conventional chromosome preparation techniques to observe and analyze the karyotypes of five natural geographic populations of Chionanthus retusus in Shandong Province, in order to reveal the karyotypic features, differences, and phylogenetic relationships among different populations, and to lay the cytological foundation for further investigation of the systematics and evolutionary relationships of C. retusus. The results showed that: (1) the chromosome number of the five populations of C. retusus was stable, with 2n=2x=46; small chromosomes were dominant, and the karyotypes were mainly composed of median centromeres (m) and submedian centromeres (sm), with the former being more abundant, and there were two types of chromosomes, 1B and 2B. (2) The karyotype formulae of the five populations of C. retusus were as follows: population B had 2n=2x=43m+3sm, population T had 2n=2x=46m, population S had 2n=2x=45m+sm, population WS had 2n=2x=44m+2sm, and population Z had 2n=2x=45m+sm. This may be related to their respective environments. (3) The asymmetry coefficients of the karyotypes of the five populations were between 54.29% and 58.00%, and the relative evolutionary levels of the populations were B > S > WS > Z > T. (4) The results of cluster analysis showed that when the genetic distance was 1, the populations T and WS and the populations B and S were first clustered together, indicating that their genetic relationships were relatively close. The study suggests that the B and S populations are more advanced in their evolutionary process, while the T population is relatively primitive, indicating that the T population (located at Mount Tais Luohan Cliff) is relatively well protected.