Nocardiosis diagnosis is a major challenge. The clinical features and radiological findings are nonspecific. Traditionally, Nocardia identification is based on colonial and microscopical morphology and biochemical tests. However, molecular biology techniques allow a better characterization of species and biotypes. PCR-RFLP of the 65-kDa heat shock protein (HSP) gene provides a rapid, sensitive, and time and labor-efficient method for this proposal. Using this technique, six of eight isolates tested were identified as Nocardia asteroides type VI. PCR-RFLP of the 65-kDa HSP gene could be very useful for determining the incidence of this pathogen in different population groups and its association with susceptibility/resistance profiles to the drugs of choice for treatment. This work is the first molecular detection of Nocardia species in Cuba.