Background We aimed to investigate the molecular basis of β-Thalassemia intermedia (TI) in the West Bank region and its management practices. Methods This was a case series multi-center study and included 51 cases of TI. DNA sequencing was used to analyze β-globin gene mutations. Common α-globin gene mutations were screened by Gap-PCR (−α 3.7 , −α 4.2 , −- MED , ααα anti3.7 ) or DNA sequencing (α2-IVS II 5 nt del). Xmn I -158 C > T polymorphisms of Gγ-globin gene was determined by RFLP-PCR. Results Seven β-globin gene mutations were observed, namely IVS-I -6 C > T, IVS-I-110 G > A, IVS-II-1 G > A, IVS-I-1 G > A, Codon 37 Trp > Stop, beta − 101 and IVS-II-848 C > A. Ten genotypes were observed. Homozygosity for IVS-I-6 accounted for the majority of TI cases with a frequency of 74.5%. The second common β-globin gene genotype was homozygote IVS-I-110 G > A (5.8%) and homozygote IVS-II-1 G > A (5.8%). The remaining seven genotypes were each detected in about 2% of patients. α-Thalassemia mutations were seen in five patients (9.8%), and included (−α 3.7 , ααα anti3.7 and α2-IVSII-5 nt del). Xmn I polymorphism was observed in four patients (7.8%), three homozygotes and one heterozygote. Conclusions Homozygosity for the mild β-globin gene IVS-I-6 allele was the major contributing factor for the TI phenotype among the study subjects. The role of Xmn I SNP and α-thalassemia mutations in ameliorating the TI phenotype was observed in few patients for each factor. The beta − 101 C > T mutation was diagnosed in one patient in homozygote state for the first time in Palestine.