Leishmania infantum infantum (LII) is one of the species that causes visceral leishmaniasis (VL) in the Old World, while L. infantum chagasi (LIC) is present in the New World. Few studies address biological differences or the behavior of these strains during infection. These parasites live inside cells of their hosts, continuously evading microbicidal mechanisms and modulating the immune responses of these cells. One of the mechanisms used by these protozoa involves the L-arginine metabolism. Understanding the differences between Leishmania species and establishing an improved murine model for study of leishmaniasis are matters of extreme importance. Thereby, the objectives of this work were to analyze the biological and molecular differences between two Leishmania infantum strains (LII and LIC) and the degree of susceptibility to infection of mice with different genetic backgrounds. The infectivity in vivo and in vitro of LII and LIC strains was evaluated in BALB/c and Swiss Webster mice, as well the NOS and ARG activities. The LII strain was more infective than the LIC strain both in vivo and in vitro. In animals infected by the LII and LIC strains, differences in NOS and ARG activities occurred. In vitro, promastigotes of LII isolated from BALB/c and Swiss Webster mice showed higher ARG activity than LIC promastigotes during the growth curve. However, no difference was observed in intracellular NO production by promastigotes of these strains. The ARG gene sequences were compared, and those of both strains were identical. However, despite the similarity, the strains showed different expression levels of this gene. It can be concluded that although L. chagasi strains are considered identical to L. infantum strains from a molecular point of view, these strains have different biological behavior.