Human brucellosis is a re-emerging zoonotic disease in Yunnan Province, China; however, the molecular epidemiological profile of circulating strains remains unclear. In this study, bacteriology, multilocus sequence typing (MLST), and core genome MLST (cgMLST) were applied to investigate the epidemiological relationships among strains. Bacteriological analysis revealed that among the 127 strains, nine were identified as Brucella melitensis ( B. melitensis ) biovar. 1 and 118 as B. melitensis bv. 3, with these strains distributed across 13 cities and autonomous prefectures. Among the patients, 70.9% were male, 87.4% were livestock-exposed farmers, and 95.3% were aged 41–80 years, with infection peaking in the summer–autumn agricultural season (70.1% of cases). The 127 B. melitensis strains were classified into two sequence types (STs): ST8 (n = 116) and ST39 (n = 11). ST8, is widely distributed in Northern China, whereas ST39 has mainly been reported in Southwestern China (Guizhou and Sichuan Provinces). These data indicate that B. melitensis sequence types ST8 and ST39 are jointly associated with the brucellosis epidemic in the human population of Yunnan Province. cgMLST analysis classified 127 strains into eight clusters (CI–VIII) and 21 STs, with 19 STs comprising strains from at least two cities isolated across two years. These findings suggest that the brucellosis epidemic in this region is driven by strains sharing a high degree of genetic homology. Global cgMLST analysis revealed high genetic similarity between the majority of strains in this study and isolates from Northern China, implying latent epidemiological links among these populations. Prioritizing dominant ST8 lineages is essential for effective brucellosis control and surveillance.
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