Hemophilia A is a bleeding disorder caused by heterogeneous mutations of the factor VIII gene. A total of 60 unique mutations have been identified in exon 16. The current study was done with the objective of detecting small mutations in exon 16 of factor VIII gene in Indian cases with hemophilia A and to further analyze structural and functional alterations in protein structure. In all, 40 cases with mild and moderate hemophilia A, negative for intron 22 inversion mutations were screened with single-strand conformational polymorphism (SSCP) for point mutations in the exon 16 region. Two cases from unrelated families showed the presence of a missense mutation due to conversion of CGT to CAT at codon 1781 in which arginine was replaced by histidine residues, resulting in deficiency in A3 domain function. Small mutation detection can be achieved using a low-infrastructure SSCP-DNA sequencing protocol in developing countries. Protein modeling predicts structural and functional changes defining causative mutations.