Alpha thalassaemia is highly prevalent in the plural society of Malaysia and is a public health problem. Haematological and molecular data from 5016 unrelated patients referred from various hospitals to the Institute for Medical Research for thalassaemia screening from 2007 to 2010 were retrieved. The aims of this retrospective analysis were to describe the distribution of various alpha thalassaemia alleles in different ethnic groups, along with their genotypic interactions, and to illustrate the haematological changes associated with each phenotype. Amongst the patients, 51.2% (n = 2567) were diagnosed with thalassaemia. Of the 13 thalassaemia determinants screened, eight different deletions and mutations were demonstrated: three double gene deletions, - - (SEA), - - (THAI), --(FIL); two single-gene deletions, -(3.7) and - (4.2); and three non-deletion mutations, Cd59G > A (haemoglobin [Hb] Adana), Cd125T > C (Hb Quong Sze) and Cd142 (Hb Constant Spring). A high incidence of -(3.7) deletion was observed in Malays, Indians, Sabahans, Sarawakians and Orang Asli people. However, the - - (SEA) deletion was the most common cause of alpha thalassaemia in Chinese, followed by the -(3.7) deletion. As many as 27 genotypic interactions showed 1023 thalassaemia silent carriers, 196 homozygous (+) thalassaemia traits, 973 heterozygous (0) thalassaemia carriers and 375 patients with Hb H disease. Statistical analysis showed a significant difference in the distribution of thalassaemia determinants amongst the various ethnic groups. Hence, the heterogeneous distribution of common determinants indicated that the introduction of an ethnicity-targeted hierarchical thalassaemia screening approach in this multi-ethnic Malaysian population would be effective.