Plasminogen deficiency is a rare disease characterized by ligneous conjunctivitis and infections. We observed a 3-year-old Italian boy presenting ligneous conjunctivitis and low plasma levels of plasminogen. Twenty-three different mutations on the PLG gene have been reported to date, but mutation analysis had been troublesome for the presence of highly homologous genes. The aim of the study was to identify the underlying mutation avoiding coamplification of unwanted genetic materials using a long polymerase chain reaction strategy, instead of the previously reported subcloning methods. By this simple strategy the complete sequence analysis of PLG gene was performed, and a previously reported missense homozygous mutation (K19E) was identified.