Cutaneous leishmaniasis in Venezuela has been associated with different Leishmania species and the visceral form is frequently related to L. chagasi. However, in this study we have characterized as L. colombiensis by molecular techniques some isolates of Leishmania from patients presenting either cutaneous or visceral leishmaniasis. Three different molecular markers (two DNA and one protein) were used to investigate the diversity among the L. colombiensis isolates. The analysis revealed the isolates to be similar although distinct from the reference strain of L. colombiensis. Slight variation among the isolates could not be correlated with any epidemiological features of the samples however the visceral isolates were observed to cluster on a phenetic tree derived from one DNA marker. The results confirm previous findings that L. colombiensis can cause both the cutaneous and visceral forms of the disease and that there exists at least two distinct etiological agents of American visceral leishmaniasis (AVL). The report also shows the necessity of careful differentiation of leishmanial isolates in any clinical or epidemiological study of human leishmaniasis.