The aim of this study was to investigate the spectrum of thalassemia mutations in Yunnan Province, Southwestern China. We detected 450 thalassemia patients and carriers by multiplex gap polymerase chain reaction (gap-PCR), PCR reverse dot-blot hybridization and direct sequencing methods in 535 suspected patients. Four types of α-thalassemia (α-thal) mutations, - -(SEA) (59.2%), -α(3.7) (rightward) (19.0%), Hb Constant Spring [Hb CS, α142, Term→Gln, TAA>CAA (α2), α(CS)α] (15.5%), and -α(4.2) (leftward) (6.34%) were detected. Six types of β-thal mutations, the most prevalent being Hb E [β26(B8)Glu→Lys, GAG>AAG or codon 26 (G>A)] (30.5%), followed by codon 17 (A>T) (20.8%), codons 41/42 (-TCTT) (17.5%), IVS-II-654 (C>T) (17.2%), -28 (A>G) (6.95%), and codons 71/72 (+A) (2.42%) were also detected. Other rare mutations were codons 27/28 (+C), IVS-I-1 (G>T), Hb New York [β113(G15)Val→Glu, GTG>GAG], Hb D-Los Angeles [β121(GH4)Glu→Gln, GAA>CAA], codon 5 (-CT), Hb G-Taipei [β22(B4)Glu→Glu (GAA>GGA)], Hb J-Lome [β59(E3)Lys→Asn (AAG>AAC)], Hb J-Bangkok [β56(D7)Gly→Asp (GGC>GAC)], IVS-I-2 (T>C), and -31 (A>C). In this study, we provide a complete mutation spectrum of α- and β-thal mutations and a valuable strategy for accurate molecular diagnostic testing in Yunnan Province, People's Republic of China (PRC).