Objective: Hemophilia B (HB) is an X-linked hereditary bleeding disorder seen in approximately one in 30,000 live male births. Clinical findings vary according to the activity level of the coagulation factor 9. More than 1,200 mutations have been identified in the F9 gene to date. Point mutations make up approximately 70% of the mutations. In this study, we aimed to determine the F9 gene mutation spectrum in HB patients in Turkey and to contribute to the genotype-phenotype relationships identifying novel mutations. Material and Methods: Fifty five patients who were followed with a diagnosis of HB in 8 different centers and molecularly analyzed in Ege University Faculty of Medicine in November 2018 and January 2020 enrolled to the study. Clinical and laboratory findings of patients were obtained from hospital records. F9 gene sequence analysis was performed using a next generation sequencing platform (MiSeq™ Illimuna) Pathogenicity of novel variants were classified according to ACMG 2015. The correlation between mutation distribution and phenotype was evaluated. Results: Among 55 HB patients enrolled in the study, severe HB phenotype were determined in 33 (60%), moderate in 15 (27.3%) and mild in 7 (12.7%). Molecular analysis was revealed 46 different variants in 54 patients (98.2%) of these variants, 30 were missense (63.8%), nine nonsense (19.1%), three frameshift (6.4%), and four splice site (8.5%) mutations. Ten of 46 variants identified has not previously been reported. In one patient no mutation was dertected by sequencing. Conclusion: In this study, the molecular diagnostic success rate and F9 gene mutation spectrum in Turkish HB patients was in accordance with the literature. The results of the study support that HB is a genotypically heterogeneous disease. Ten novel mutations were identified for the first time with this study and contributed to the genotype-phenotype relationship sof the disease.