Babesia (B.) ovis is an intra-erythrocytic protozoan parasite that infects small ruminants globally, causing economic losses. This study aimed to investigate the molecular prevalence of B. ovis in 1200 asymptomatic goats of various breeds across four districts in Punjab, Pakistan: Layyah, Lohdran, Dera Ghazi Khan, and Rajanpur. The enrolled goats represented ten breeds, including Daira Din Pannah (n = 890), Pahari goat (n = 68), Nukri (n = 44), Teddy (n = 37), Lail Puri (n = 36), Beetal (n = 36), Dessi (n = 32), Makhi Cheena (n = 27), Muhammad Puri (n = 19) and Fazil Puri (n = 11). The hematological and biochemical profiles of the goats, risk factors associated with the infection, and the phylogenetic relationship of the detected isolates were also evaluated. In total, 105 blood samples (9.6 %) tested positive by PCR. Sanger sequencing of a partial fragment of the 18S rRNA gene confirmed B. ovis. Phylogenetic analysis of the 18S rRNA gene sequences revealed 99–100 % similarity with isolates previously reported from Iran, Iraq, Turkey, and Spain. The infection rate varied across districts, with the highest prevalence observed in goats from Rajanpur (13 %), followed by Dera Ghazi Khan (11 %), Layyah (7 %), and Lohdran (5 %) (P = 0.003). The susceptibility to infection varied among goat breeds, with Lail Puri breed showing the highest susceptibility (P = 0.03). Risk factor analysis revealed that goats under one year of age and those kept on farms where other animals and dogs were also present had higher B. ovis infection rates. Babesia ovis-infected goats showed reductions in white and red blood cells, hemoglobin concentration, and alterations in serum aspartate aminotransferase and creatinine levels. This study provides updated data on the prevalence of B. ovis in local Pakistani goat populations, emphasizing the need for integrated control strategies against this tick-borne pathogen.