Mycoplasma synoviae (MS) has a major financial impact on poultry production since it can cause infectious synovitis, respiratory symptoms, and eggshell apex abnormalities in both turkeys and chickens. The current study aims to identify MS using bacteriological and molecular methods from cases suffering from arthritis in broilers and layer hens in certain Egyptian governorates. Between January 2020 and March 2022, one hundred twenty-five samples from 25 flocks/governorate representing 15 broiler chickens and 10 laying hens were from 5 Egyptian governorates (Giza, Cairo, AL-Dakahlia, AL-Qaliobia and AL-Fayoum. Conventional techniques were used to characterize the isolates, as well as 16S rRNA, and VLha specific MS primers were used for molecular identification by using PCR. Microscopic fried egg colony examination on the bacterial isolation samples showed 67/125 (53.6%) were suspected MS positive, and 58/125 (46.4%) were MS   negative. Out of   the 67 only 55 isolates were recognized as MS by conventional polymerase chain reaction (PCR) 55/67 (82%) with total rate of 55/125 (44%). Our research concluded that MS continues to be a significant avian pathogen, needed for immediate and regular monitoring of MS in chicken flocks, as well as future research to identify the best preventative and control approaches for this disease.