Macular corneal dystrophy (MCD) is an autosomal recessive disease characterized by corneal opacities and caused by mutations in a carbohydrate sulfotransferase gene, known as CHST6. MCD type I patients show missense mutations in the CHST6-coding region, and MCD type II patients show a large deletion and replacement in the upstream region of CHST6. The objective of this study was to identify the genetic defect in CHST6 gene causing MCD in Italian families. We investigated MCD genotype by using polymerase chain reaction followed by direct sequencing, and results were confirmed by restriction analysis. An enzyme-linked immunosorbent assay was performed to assess the presence of sulfated keratan sulfate in the serum of MCD patients. Biochemical analysis revealed a MCD type I phenotype in two families and a type II phenotype in another family. Two novel missense mutations and a polymorphism in the coding region of CHST6 gene were identified in patients with MCD type I. In one MCD II family, a homozygous deletion in the upstream region of CHST6 gene was found.