Three cases of nosocomial malaria are reported from patients of the Internal Medicine Ward of a tertiary University teaching hospital in Medellin, Colombia. Epidemiological research, based on entomological captures, medical records review and interviews of nursery staff about patient care practices potentially involving contact with blood, were carried out. Molecular characterization of Plasmodium falciparum was based on the amplification of MSP1, MSP2 and GLURP genes. This method enabled confirmation of the same P. falciparum genotype in all three patients as well as in a fourth one (index case). The presence of nosocomial malaria was confirmed and it was concluded that the most likely source of transmission was through multi-dose preparations of heparin applied to heparin locks.