Objectives The aim of this study was to investigate the geographic distribution of beta-globin gene mutations in different ethnic groups in Yunnan province. Methods From 2004 to 2014, 1,441 subjects with hemoglobin disorders, identified by PCR-reverse dot blot and DNA sequencing, were studied according to ethnicity and geographic origin. Haplotypes were examined among 41 unrelated thalassemia chromosomes. Results Eighteen beta-thalassemia mutations and seven hemoglobin variants were identified for 1,616 alleles in 22 different ethnic groups from all 16 prefecture-level divisions of Yunnan. The prevalence of beta-thalassemia was heterogeneous and regionally specific. CD 41-42 (-TCTT) was the most prevalent mutation in the populations of northeastern Yunnan. CD 17 (A > T) was the most common mutation in the populations of southeastern Yunnan, especially for the Zhuang minority, whereas Hb E (CD 26, G > A) was the most prevalent mutation in populations of southwestern Yunnan, especially for the Dai minority. Among the seven types of haplotypes identified, CD 17 (A > T) was mainly linked to haplotype VII (+ - - - - - +) and IVSII-654 (C > T) was only linked to haplotype I (+ - - - - + +). Conclusion Our data underline the heterogeneity of beta-globin gene mutations in Yunnan. This distribution of beta-globin mutations in the geographic regions and ethnic populations provided a detailed ethnic basis and evolutionary view of humans in southern China, which will be beneficial for genetic counseling and prevention strategies.