A total of 120 superior trees of Michelia macclurei from five natural populations in southeastern Guangxi were used as experimental materials to investigate phenotypic variation, genetic diversity, and population genetic structure, and to identify trait-associated markers. Thirteen phenotypic traits were measured, and genotyping was performed using ten simple sequence repeat (SSR) primer pairs. A mixed linear model (MLM) was further used for association analysis. Significant geographic differentiation in phenotypic traits was observed among M. macclurei populations in southeastern Guangxi. The BL population showed significantly larger leaves and higher biomass, whereas the CW population showed higher malondialdehyde and proline contents, indicating stronger stress resistance. The YL and LW populations showed the most similar phenotypic characteristics. Molecular genetic analysis showed that M. macclurei germplasm resources in southeastern Guangxi possessed abundant genetic diversity, with a mean polymorphism information content of 0.62, although heterozygote deficiency was generally detected. Among the five populations, the YL population had the highest genetic diversity, whereas the high-altitude LW population had the lowest. The genetic differentiation coefficient among populations was 0.11, and gene flow reached 2.89, indicating frequent genetic exchange. Genetic structure analysis showed a complex genetic background among populations, and the BL and YL populations retained relatively independent genetic components. Association analysis detected five SSR loci significantly associated with phenotypic traits, with explained variation ranging from 6.87 percent to 18.90 percent. Among them, locus M23 showed significant pleiotropic effects. This study clarifies the genetic basis of M. macclurei in southeastern Guangxi. The identified associated loci, such as M23 and M25, provide a scientific basis for germplasm conservation and marker-assisted breeding of M. macclurei.