Chlamydia trachomatis causes conjunctivitis to spread throughout the world, it infects people of all ages and can cause blindness if not treated properly. Therefore, the current study aimed to diagnose C. trachomatis that causes conjunctivitis and identify the detection of the aac(3)-I gene in C. trachomatis isolated from conjunctivitis by using Polymerase chain reaction (PCR ) technique. One hundred and seven swabs were collected from patients infected with conjunctivitis from different ages (1 month –70 years) and for both sexes (63 samples from females and 44 samples from males) for the period from 27 November till 19 May 2023 from Al-Najaf province in Iraq. Purulent material from the conjunctiva sac was collected on a sterile swab and placed in phosphate-buffered saline and incubated in a deep freezer after completing the collection. All samples were subjected to a PCR to detect the major outer membrane protein (MOMP) gene to diagnose the Chlamydia trachomatis. where the results showed that out of 107 samples, 26 (24.3%) were positive for C. trachomatis. The individuals infected with C. trachomatis were categorised into multiple age groups. The age group ranging from one month to 14 years exhibited the highest prevalence of conjunctivitis. The age group between 15-28 years was followed by the age group between 57-70 years, while the lowest group for conjunctivitis was the age group between 43-56. The existence of the aac(3)-I gene, which confers resistance to the antibiotic gentamicin routinely employed for the treatment of eye infections, was identified. Among the 26 isolates of C. trachomatis, 15 (57.69%) were found to have the aac(3)-I gene. The present investigation demonstrated the significant involvement of C. trachomatis in conjunctivitis, with a higher susceptibility observed among women. The prevalence of conjunctivitis was higher in the age range of 1-28 compared to other age groups. The present investigation exhibited the dissemination of the gentamicin resistant aac(3)-I gene among C. trachomatis bacteria.