Mycoplasma is a significant microorganism of shelter cats, which can cause respiratory infections and conjunctival inflammation, bringing about huge financial and health misfortunes to pet cats worldwide. This study was undertaken to investigate the prevalence and anatomical distribution of mycoplasmas in a population of shelter cats and establish the association of their presence with ocular or respiratory infections in shelter cats in Baghdad province. A total of 450 swabs were collected, including nasal, oropharyngeal, and conjunctival swabs 150 each from shelter cats of different ages, sex, and breed. The swabs were cultured in PPLO agar, and incubated at 37°C for 2 weeks, and the growing colonies with dissecting microscope. The colonies were also stained with Dienes stain. The prevalence of Mycoplasma in all examined cats was 40.7%, as determined by culture. The findings revealed that the prevalence of Mycoplasma in upper respiratory tract infections in female cats older than one year was between 46.9% and 48.7%. Conversely, the infections exhibited greater prevalence and a higher rate of isolation in males under one year of age 28.8-30.8%. The present investigation highlighted a significant prevalence of Mycoplasma in respiratory swabs obtained from Persian and Himalayan cats, but Scottish and British cats exhibited a comparatively lower rate of positive Mycoplasmal culture. To conclude, Mycoplasmal infections were more prevalent in upper respiratory diseases among shelter cats.