Introduction Thalassemia is the most common monogenic disease in South and Southeast Asia. An accurate assessment of the relative frequency and composition of thalassemia mutations is important for the design of appropriate strategies to prevent the disease. In this study, we aimed to decode the molecular characterization of thalassemia mutations in Zhuhai region of southern China. Methods A total of 8048 individuals who were potential thalassemia carriers were enrolled. Gap‐polymerase chain reaction (Gap‐PCR) and reverse dot‐blot (RDB) hybridization methods were employed to detect common deletional and non‐deletional thalassemia mutations. Multiplex ligation dependent probe amplification (MLPA) and Sanger sequencing were used to analyze and verify rare and complex mutations. Results We diagnosed 3433 individuals as thalassemia carriers or patients. Of these, 2395 (69.76%) individuals with α‐thalassemia harbored 13 α‐globin gene mutations. The three most common α‐thalassemia mutations were ‐‐SEA (60.08%), ‐α3.7 (20.62%) and ‐α4.2 (9.25%). We diagnosed 903 (26.30%) individuals with β‐thalassemia and identified 20 β‐globin gene mutations, of which the three most frequent were CD41/42 (‐TCTT) (38.10%), IVS‐II‐654 (C>T) (23.69%) and TATAbox‐28 (A>G) (15.18%). In addition, we identified 15 rare thalassemia variants. We also summarized the association between the thalassemia genotype and hematological parameters, which demonstrated the broad phenotypic heterogeneity caused by globin gene mutations. Conclusion This is the first survey of thalassemia molecular epidemiology and hematological phenotype in Zhuhai region. It uncovered a high prevalence and complex molecular spectrum of thalassemia. These findings can be used as a basis for thalassemia diagnosis, counseling and prevention management.