One hundred and sixty clinically diseased broiler local chickens collected from 22 different farms belonging to Qalubia, Dakahlia and Gharbia governorates were subjected to bacteriological examination and molecular characterization. Clinical Signs were chronic respiratory disease with respiratory manifestations,lameness, loss or reduction in egg production.Bacteriological examination showed that 18.18% of the isolates were Mycoplasma positive and showed growth in pleuropneumonia-like organism (PPLO) agar plates, with fried egg appearance when examined by stereoscopic microscope. Mycoplasma colonies were tested for antimicrobial sensitivity tests against 12 antimicrobial antibiotics and showed higher sensitivity to nitrofurantoin, gentamicin, norfloxacin, ciprofloxacin and neomycin. Intermediate sensitivity were recorded to ceftriaxone and cefotaxime and antibiotic resistance were recorded to tetracyclines, lincomycin, chloramphenicole and sulphamethoxasine. Identification of the bacterial strains of the isolates were conducted by multiplex PCR by using two primer pairs for Mycoplasma synoviae (vlhA) and Mycoplasma gallisepticum (mgc2). The amplicons expected size were 396 bp, and 300 bp for vlhA and mgc2 respectively. Only13 out of 22 farms were positive, representing 59.09 %.Moreover, the incidence rate of Mycoplasma synoviae (vlhA) and Mycoplasma gallisepticum (mgc2) were 22.72% and 13.63% respectively and 22.72% of the inspected farms showed positive results for both Mycoplasma strains.