Bovine mastitis caused by Mycoplasma bovis represents a major problem for dairy industry all over the world. Although Mycoplasmas were identified in Egypt decades ago, the prevalence of Mycoplasma bovis mastitis is not frequently investigated. The current study was designed to monitor the prevalence of Mycoplasma bovis in clinical bovine mastitis milk in Egypt. Clinical mastitis milk samples (n=703) were collected between 2016 to 2019 from different dairy farms located in different governorates in Egypt including, Giza (227), Alexandria (357), Dakahlia (78), Buhayrah (27), Ismailia (14) and presented for Mycoplasma isolation using conventional cultural method followed by molecular identification of Mycoplasma bovis using PCR targeting mb-mp 81 gene of M. bovis. From the examined samples (n=703), Sixty-three (8.96%) were positive for Mycoplasma isolation. Among the 63 Mycoplasma isolates, 53 were identified by PCR as Mycoplasma bovis representing 84.12% of the recovered Mycoplasma isolates (n=63) and 7.53% from the total examined mastitis milk samples (n=703). From the 53 Mycoplasma bovis isolates, 17/227 (7.48%) were obtained from Giza, 13/357 (3.64%) from Alexandria, 18/78 (23.07%) from Dakahlia, 1/27 (3.70%) from Al-Buhayrah and 4/14 (28.57%) from Ismailia.