Objective: Cutaneous leishmaniasis (CL) has recently grown into a major public health problem in 88 countries of the world, including Iran. It is a polymorphic disease which may show various clinicopathological features. Although the effect of the genetic diversity of the parasite has been demonstrated as one of the factors influencing clinical manifestations in CL, no data exist regarding the genetic variation of Leishmania major and its microscopical features. Study Design: Fine-needle aspiration, touch smears and the histological sections of 100 patients were examined for Leishmania amastigotes, using Giemsa and hematoxylin and eosin. Diverse types of inflammatory cells in the 40 positive and 5 negative smears were differentiated. Kinetoplast DNA (kDNA) was amplified using nested PCR and subsequently sequenced. Sequencing analysis of the amplified kDNA was used to investigate the genetic variations among L. major isolates and to correlate the findings with microscopical features and geographical origins. Results: The quantified amastigote density in the 40 positive touch smears was blindly classified by 3 observers. Grade I, II, III and IV had 7, 13, 9 and 11 cases, respectively. The microscopical features, the mean percentage of neutrophils, lymphocytes and other inflammatory cells, and the leishmanial density of the grades and negative cytopathological samples were contrasting. kDNA amplification of L. major was detected from the cutaneous lesion, and 21 of these amplicons were successfully sequenced. Conclusions: These results indicate that L. major strains causing CL in southern Iran are genetically diverse; furthermore, a correlation between the genetic heterogeneity of the parasite, the microscopical manifestation and the geographical regions of the disease in humans was found.