Toxoplasma gondiiis a globally significant parasite that affects various vertebrates, including livestock, posing health risks and economic losses, particularly in poorly managed regions. A total of 450 blood specimens were randomly collected from goats (Capra hircus) of Malakand Division, Khyber Pakhtunkhwa, Pakistan. Genomic DNA was extracted, and a partial fragment of the Toxo-B1 gene was amplified and sequenced. The obtained sequences were aligned with homologous sequences retrieved from NCBI for phylogenetic analysis. The overall prevalence of T. gondii infection in goats was 7.3% (33/450). The infection rate significantly differed between treated goats (3.4%, 7/207) and untreated goats (10.7%, 526/243) (P = 0.003). Analysis of drinking water sources showed varying prevalence rates: bore water at 1.9% (2/107), open water at 9.6% (22/228), tap water at 12.5% (4/32), and drain water at 6% (5/83) (P = 0.046). Nucleotide BLAST analysis of the partial sequences of the Toxo-B1 gene showed 99.30% identity with sequences from humans, wild rats, and domestic chickens. Phylogenetic analysis linked our T. gondii isolates from goats to those reported from Poland, Iran, and Nigeria. This study underscores the necessity for enhanced management and sanitation practices to mitigate T. gondii transmission in goats. Regular surveillance and molecular characterization are crucial for understanding the epidemiology of T. gondii and reducing its impact on livestock health and agricultural productivity.