The clinical expression of zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania (L.) major parasites has a broad spectrum ranging from asymptomatic infection to self-limited cutaneous sores or severe disease. In concert with the host immune responses, the vector variability and the number of bites, genetic variation between L. major isolates might impact on the clinical output of the disease. We investigated herein the intra-specific variability of L. major field isolates independently of host or vector factors and then tried to correlate parasite variability to ZCL severity in corresponding patients. Several assays were applied, i.e., in vivo pathogenicity of promastigotes in a BALB/c mice model, resistance/sensibility to complement lysis, in vitro growth kinetics, and expression of different lectins on the promastigote surface. Combining all these parameters allowed us to conclude that the resistance to complement lysis and PNA/Jacalin lectins binding to parasite surfaces are important markers of parasite virulence. These factors correlate significantly with clinic polymorphism of ZCL and modestly with genetic micro-heterogeneity, a characteristic we previously revealed with a MLMT profile.