Human Leishmania infantum infection results in a spectrum of clinical expressions ranging from cutaneous to either asymptomatic or fatal visceral disease. In this context, characterization of parasite virulence appears to be relevant as a biological marker of intrinsic parasitic factors that can affect the pathology of leishmaniasis. Since parasite populations in naturally infected hosts are likely to be composed of multiclonal associations, we first explored the biodiversity of parasite virulence at the intrastrain level in vitro and in vivo by using 11 clones isolated from three strains previously known to express different virulence phenotypes in mice. Subsequently, we studied the course of infection in mice inoculated simultaneously or successively with strains or clones showing various virulence phenotypes. Analysis of in vitro growth characteristics showed no differences among clones from the different parental strains. By contrast, in vivo experiments evidenced a marked intrastrain heterogeneity of virulence to mice. One out of five clones obtained from a virulent strain showed a typical virulence phenotype, while the remaining four clones had low-virulence profiles, as did the six clones isolated from two low-virulence strains. In mixed multiclonal infections, the virulence phenotype was expressed as a dominant character over the associated low-virulence clones. After a challenge with either a homologous or a heterologous strain or clone, virulence phenotypes were conserved and expressed as in naive mice independently from the preexisting population. These results strongly suggest that parasite virulence in L. infantum visceral leishmaniasis is clonal and dominant in nature.