Background:  Microsporum canis (M.canis) is the most common dermatophyte causing tinea capitis and corporis in humans. Proteases encoded by subtilisin (SUB) virulence genes are from the keratinases leading to dermatophytosis. Subtilisin 1 and SUB3 were expressed during the adherence of arthroconidia to corneocytes by M. canis. Few M. canis molecular identification assays were used. There is a new target gene for identification of M.canis such as the velB gene. The aim of our study is to assess the presence of SUB (1-3) virulence genes in M.canis isolates and to assess the ability of the velB gene in identification of M.canis isolates compared with the traditional Mc gene. Methods: Thirty M.canis isolates were collected from 120 suspected cases of dermatophytosis attended the Dermatology outpatient clinic in Suez Canal University Hospitals. Polymerase chain reaction (PCR) for detection of MC gene was done. Real time PCR was done for detection of the velB and SUB (1-3) genes. Results: The number of positive isolates detected of MC gene were 28(93.3%) isolates. Thirty isolates (100%) were positive for velB gene. Subtilisin 1 gene was detected in 21 isolates (70%), while SUB 2 gene was positive in 3 isolates only (10%) and SUB 3 gene showed the highest percentage as it was positive in 29 isolates (96.7%). Conclusion: We concluded that the new primer Mc-VelB showed high specific results in detection of M.canis isolates. Subtilisin genes 1- 3 were detected with high percentage in our study that proves having a major role in the infection by M.canis.