Johne’s disease (JD), chronic granulomatous disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), is regarded as a potential public health issue and a significant threat to the dairy and agricultural food businesses. This study was conducted for the molecular investigation of MAP in cattle reared for small-scale dairy production in Sharkia Governorate, Egypt. Seventy-five fecal samples were collected from diarrheic and healthy dairy cows that came into contact with diarrheic ones for molecular screening. Clinically, 32 of 75 examined cattle showed variable degrees of body weight loss and diarrhea. The PCR targeting the insertion sequence IS900 gene revealed that 22.6% of examined cattle were positive for MAP. A rate was significantly higher in diseased older cattle than in young cattle with no symptoms of the disease. Furthermore, DNA sequencing and phylogenetic analysis revealed that our strains (ON816021 and ON816022) were 100% identical showed complete identity with MAP-C (M. paratuberculosis of cattle origin) as Japanese (CP066812) and German strain (CP022105). Overall, the findings highlight the potential of the IS900 gene-based molecular technique for MAP detection in small dairy cattle farms.