Ticks are hematophagous ectoparasites of terrestrial and semi-aquatic vertebrates that may transmit microor-ganisms to their hosts. Spirochetes of the genus Borrelia are common in ticks and an incipient group has been identified in association with reptiles and their tick parasites. To overcome the knowledge deficit, this study aimed to morphologically and molecularly identify ticks infesting wild lizards and to molecularly assess Borrelia spp. associated with these ticks in Pakistan. For this purpose, free-ranging monitor lizards (Varanus bengalensis) from Khyber Pakhtunkhwa province, Pakistan, were examined for tick infestations. A total of 776 ticks were collected from 36/63 lizards, resulting in a prevalence of 57% (95% CI 44.7-69.3%), overall mean intensity of 21.5 (95% CI 18.9-24.1) ticks per infested lizard, and overall mean abundance of 12.3 (95% CI 9.25-15.4) ticks per examined lizard. All ticks were morphologically identified as Amblyomma gervaisi. The morphological identification of the ticks was molecularly confirmed through sequencing fragments of the mitochondrial 16S rRNA gene. In addition, a fragment of nuclear second internal transcribed spacer (ITS2) was generated for the first time for A. gervaisi. Phylogenetic analysis inferred from tick 16S rRNA gene partial sequences predicted a close evolutionary relationship of the collected A. gervaisi ticks with conspecific sequences from India, which shared 94.5% identity. Through two PCR assays targeting fragments of the borrelial genes, 16S rRNA and flaB, 19 (18%) out of 108 ticks yielded borrelial DNA. Phylogenetic analyses inferred from DNA sequences of the two borrelial genes revealed that the Borrelia sp. from A. gervaisi detected in this study belonged to the reptile -associated Borrelia group (REP). This is the first molecular report of ticks infesting monitor lizards and associ-ated Borrelia sp. in Pakistan. The preliminary phylogenetic analyses of A. gervaisi may assist in understanding the molecular epidemiology of Amblyomma spp.