Boophilus microplus, an ectoparasite of bovines has immense importance in South Asian countries like Pakistan. These are cosmopolitans and affect cattle herds drastically worldwide and are involved in direct and indirect harm to the bovine species. In this regard, trichlorfon is widely used to control ticks in bovines along with its sprays to control different pests, which leads to resistance in ticks against these acaricides in different parts of the world. Therefore, the present study aims to investigate the resistance and prevailing genomic mutations in Boophilus microplus ticks against trichlorfon. Molecular characterization was carried out and heterozygous mutations were observed in the adult tick samples collected from two different zones of the Khyber Pakhtunkhwa (KPK) province of Pakistan. After the identification of ticks, in vitro studies were performed which showed resistance in ticks against trichlorfon drug. To assess genomic mutation in the samples, DNA was extracted through a kit, and PCR was performed to evaluate the mutation in the samples. After in-vitro studies, almost 104 (54%) ticks samples were found resistant and run through PCR in which 3 and 4 samples from zone-I and zone-II, were identified mutated while the remaining samples were found non-mutated. Boophilus microplus acetylcholinesterase genes were sequenced and compared with the control group. All these findings suggest a continuous monitoring of heterozygous mutations in the Boophilus microplus ticks and the strategic application of trichlorfon against Boophilus microplus ticks, to enable improvement in drug efficacy and tick control strategies.