Piroplasmosis is one of the most important diseases of livestock, constraining optimal production and leading to economic loss. This study was carried out to detect Theileria annulata using 2 different molecular techniques: recombinase polymerase amplification (RPA) and conventional PCR. Blood samples were collected from 274 ticks infesting asymptomatic cattle from several counties of Chakwal, Faisalabad, and Jhang districts of Punjab province of Pakistan using FTA cards. Following extraction of genomic DNA, each sample was subjected to RPA optimized to amplify a 281 bp fragment of the Enolase gene for T. annulata. Specificity of the test was confirmed using positive DNA samples of related piroplasm species, while analytical sensitivity was calculated using different serial dilutions of a long fragment of the same gene. The RPA results were positive for 48 of 274 (17.51%) samples. All 274 samples were screened using conventional PCR and 21 (7.66%) samples were positive for T. annulata. All the samples that were RPA positive but PCR negative were sequenced, which confirmed the results of RPA. The highest positive rate was found in Chakwal district, followed by Faisalabad and Jhang. This study demonstrates the application of highly sensitive and specific rapid diagnostic methods for T. annulata to a regional screening program. This is the first report of tick-borne disease from Pakistan using RPA.