Haemophilia B is an X-linked recessively inherited disease caused by highly heterogeneous mutations in the factor IX gene. Very few studies are available on the nature of mutations in haemophilia B patients from India. The present study was undertaken with an aim to characterize the mutations present in haemophilia B patients from western India. The screening for mutations was done by conformation sensitive gel electrophoresis which was subsequently confirmed by DNA sequencing. A total of 57 mutations were detected in 93 haemophilia B patients - 45 single-base substitutions, one a donor splice, two small deletions and nine nonsense mutations. Seventeen novel mutations and two common mutations were also detected in the present study. Majority of the mutations were located in the exon h of the factor IX gene which codes for the catalytic domain. The spectrum of mutations in haemophilia B patients reported from other parts of India has been compiled and a comprehensive analysis has been presented.