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Background: Pattern recognition receptors (PRRs) as the recognition of pathogenic fungal structures induce the secretion of cytokines by immune systems. Toll‐like receptors (TLRs) 2 and 4 are the main PRRs that recognize fungal components. Aim: The present study aimed to assess the presence of dermatophyte species in symptomatic cats in a region of Iran and to investigate the expression of TLR‐2 and 4 in cat lesions with dermatophytosis. Methods: A total of 105 cats suspected of dermatophytosis with skin lesions were examined. Samples were analysed by direct microscopy using potassium hydroxide (20%) and culture on Mycobiotic agar. Dermatophytes strains were confirmed by the polymerase chain reaction (PCR) amplification and then sequencing of the Internal Transcribed Spacer rDNA region. Also, for pathology and real‐time PCR studies, skin biopsies were taken by sterile single‐use biopsy punch from active ringworm lesions. Results: Dermatophytes were found in 41 felines. Based on the sequencing of all strains, Microsporum canis (80.48%, p < 0.05), Microsporum gypseum (17.07%) and Trichophyton mentagrophytes (2.43%) were the dermatophytes isolated from cultures. Cats under 1 year (78.04%) revealed a statistically significantly higher prevalence of infection (p < 0.05). Gene expression by real‐time PCR revealed the increased TLR‐2 and 4 mRNA levels in skin biopsies of cats with dermatophytosis. Conclusions: M. canis is the most prevalent dermatophyte species isolated from feline dermatophytosis lesions. Increased expression of TLR‐2 and TLR‐4 mRNAs in cat skin biopsies suggests that these receptors are involved in the immune response by recognizing dermatophytosis. [ABSTRACT FROM AUTHOR]