Humoral (antibody) response is an important part of immunity against pathogens. Despite the clear role of cell-mediated immune response in protection against leishmaniasis, the role of humoral responses is challenging. There is very limited data regarding humoral immune response against Leishmania tropica which is the causative agent of human cutaneous leishmaniasis in many parts of the world. Here, we have compared pathogenicity and antibody response against six Iranian Leishmania tropica isolates in BALB/c mice. A Leishmania major isolate was used for comparison. The parasites were injected into the mice followed by the evaluation of the lesion development, parasite load, and antibody responses (IgG1 and IgG2a). Our findings showed that some isolates caused the large lesions and high parasite load in the spleen and lymph node, while other isolates led to no lesion, no splenic parasitism, and low parasite load in the lymph node. The more pathogenic isolates induced higher antibody responses (IgG1 and IgG2a). Our results indicated that there is substantial heterogeneity among various Leishmania tropica isolates regarding the humoral immune response as well as the pathogenicity.