Leishmania infantum has been described as a highly polymorphic group of parasites, responsible for visceral leishmaniasis and cutaneous leishmaniasis. In this paper we report the life-cycle of L. (L.) infantum in an endemic area of visceral leishmaniasis in Venezuela, by using molecular diagnosis and characterization of parasites isolated from dogs, humans with visceral leishmaniasis and sand flies. The molecular characterization was carried out by use of kDNA restriction analysis, dot-blot hybridization with species-specific probes and RFLP of the PCR products. The results demonstrated that L. (L.) infantum is the parasite responsible for VL in the island. The parasites were revealed to be genetically homogeneous with no intra-specific differences between isolates from different individuals. The highest homology of the isolates was with L. (L.) infantum from the Old World rather than with L. (L.) chagasi from the New World. Additionally, we report the geographical distribution of Lutzomyia longipalpis, and the relationship with the transmission of L. (L.) infantum in the studied area.