Sickle cell disease has been a major inherited blood disorder in several populations of the world including Tharu tribal communities of Nepal and India. This cross sectional study aims to access the prevalence of sickle cell disease in Tharu communities of Nepal by three different tests.Total 386 blood samples were collected from eight different sub-ethnic groups of Tharu communities of Rupandehi district, Nepal. The samples were tested for presence of sickle haemoglobin by sickle cell solubility test and sickling test as screening test method and those seen positive from either of these two tests were undertaken through genomic test by restriction fragment length polymorphism. Out of total 386 samples, 67 were seen positive from sickling test and 68 from solubility test. One and two samples were positive for only sickling test and solubility test respectively. 69 samples were tested by molecular method for confirmation and genotyping and out of them two were wild, 57 hetero and 10 mutant. Kanwar Tharu had highest prevalence(53.94%) and Marchaha Tharu had lowest prevalence(0%) of sickle cell allele in their gene pool. The allelic frequency of HbA and HbS were found to be 0.9 and 0.1 respectively. Prevalence of sickle cell anaemia was not found to be common in Tharu community, χ2(2, N=386)=686.389, p˂ .00001). The distribution of sickle cell allele was cast dependent, χ2(7, N=386)=58.2823, p˂ 0) and Kanwar Tharu and Dadaha Tharu are most severely affected sub-ethnic groups with about 50% occurrence of HbS in their population.