Q fever is an infectious disease of animals and humans, caused by globally distributed C. burnetii. In Iraq, there are no previous studies associated with the detection of the organism in cattle. An overall of 130 lactating cows were submitted to direct collection of milk samples. Initially, the samples of milk were tested using the molecular polymerase chain reaction (PCR) assay targeting three genes (16S rRNA, IS1111a transposase, and htpB). However, positive results (18.46%; 24/130) were detected only with the 16s rRNA gene. Concerning risk factors, the highest prevalence of C. burnetii was showed in the district of Badra (42.86%), whereas the lowest - in Al-Numaniyah and Al-Suwaira districts (P=0.025). There was no significant variation in positivity between the months of sampling period (P=0.082) and between age groups (P=0.076). Crossbred cows (20.69%) showed a higher positivity than local and pure breeds (P=0.043). Milk of positive samples (n=24) was used for cultivation of C. burnetii into specific pathogen free-embryonated chicken eggs (SPF-ECEs). After three passages into SPF-ECEs, contents of yolk sac were collected, subjected for DNA extraction, and re-tested by PCR assay using the primer of 16s rRNA gene only. Of 24 cultivated milk samples, 12.5% (3/24) were positive for C. burnetii. Finally, the positive local isolates were analysed phylogenetically and reported in NCBI-Genbank under the accession numbers of MN121700.1, MN121701.1, and MN121702.1. In conclusion, this is a unique study as it detected C. burnetii in Iraqi lactating cows, and confirmed that organism was shed actively through milk, suggesting that these animals can play a role as a reservoir for organism with potential risk for transmission of infection from these animals to humans as well as to other animal species.