A complete cDNA sequence is reported for LRV2-1, the first Leishmania RNA virus known to infect an Old World parasite, Leishmania major. Sequence analyses show that LRV2-1 differs significantly from members of the LRV1 genus which infect New World parasites. The data support a view that transmission of LRV is strictly vertical and suggest that LRV predate the divergence of Old and New World parasites. As a consequence of this divergence, conserved features can be identified for the first time in Leishmaniavirus proteins. A finding that the virus capsid and polymerase genes do not overlap is unique among the known Totiviridae and infers that a gag-pol fusion protein cannot be produced simply via tRNA slippage in LRV2-1.