Coxiella burnetii, the etiological agent of Q fever, is a globally significant zoonotic pathogen posing substantial threats to both public health and the livestock industry. Despite its widespread distribution, comprehensive molecular epidemiological data regarding this pathogen in Iraq remain scarce. This study aimed to directly detect, determine the prevalence, and genetically characterize C. burnetii in ticks infesting apparently healthy sheep in the Dhi Qar province of Iraq. A total of 400 tick specimens were collected from sheep across various regions of the province. Following species identification, the ticks were snap-frozen in liquid nitrogen and pulverized using a sterile scalpel. Genomic DNA was extracted from the powdered samples using a commercial extraction kit. Subsequently, molecular detection of C. burnetii was performed using specific primers targeting the IS1111 gene, employing a nested polymerase chain reaction (nested-PCR) assay to maximize diagnostic sensitivity and specificity. The results revealed an overall C. burnetii infection rate of 17.25% among the examined ticks. Statistical analyses indicated that the prevalence of infection was significantly higher in ewes and sheep older than three years compared to lambs and males. This disparity likely reflects the gradual accumulation of the pathogen or prolonged exposure to contaminated environments over time. As the first molecular report documenting the presence of C. burnetii in sheep ticks in Dhi Qar province, this study serves as a critical alert for public health. These findings underscore the urgent need to implement active surveillance programs, enhance tick control measures, and raise health awareness in rural communities across Iraq to mitigate the further spread of this zoonotic disease.