Brucellosis is one of the most widespread zoonotic diseases that causes a great economic loss in most countries, particularly in low- and middle-income countries, and those where no eradication programs are applied. The study covered the period of 6 months (Started from February to July 2021) to investigate the prevalence of brucellosis in sheep slaughtered in three districts slaughterhouses of Al Muthanna governorates. A total of 315 blood samples of slaughtered sheep of both sexes (114 males, 201 females), were examined usingof Rose Bengal cart test (RBT), results indicated that, out of all samples tested, (12) of both sexes were found positive reactors (3.80%) including (9) females (2.85%) and (3) males (0.95%). The infection rate of this zoonotic disease varied among the studied districts in the governorate, hereby, the present result represents Al Khudhur, district which recorded the highest infection rate (4.55%) followed by Al Rumaitha district (3.64%), whereas Al Samawah district recorded the lowest prevalence (2.61%). The present calculated results could establish a foundational data base regarding the occurrence of this diseasein the sheep population of Al Muthanna governorates. The current results of the study recommend the need for more detailed study covers the incidence of brucellosis in large samples of sheep annually and isolation of the local bacterial strains in Al Muthanna governorates.