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<i>Rhipicephalus microplus</i> tick highly affects the veterinary sector throughout the world. Different tick control methods have been adopted, and the identification of tick-derived highly immunogenic sequences for the development of an anti-tick vaccine has emerged as a successful alternate. This study aimed to characterize immunogenic sequences from <i>R. microplus</i> ticks prevalent in Pakistan. Ticks collected in the field were morphologically identified and subjected to DNA and RNA extraction. Ticks were molecularly identified based on the partial mitochondrial <i>cytochrome C oxidase subunit</i> (<i>cox</i>) sequence and screened for piroplasms (<i>Theileria/Babesia</i> spp.), <i>Rickettsia</i> spp., and <i>Anaplasma</i> spp. PCR-based pathogens-free <i>R. microplus</i>-derived cDNA was used for the amplification of full-length cysteine protease inhibitor (cystatin 2b), cathepsin L-like cysteine proteinase (cathepsin-L), glutathione S-transferase (GST), ferritin 1, 60S acidic ribosomal protein (P0), aquaporin 2, ATAQ, and <i>R. microplus</i> 05 antigen (Rm05Uy) coding sequences. The <i>cox</i> sequence revealed 100% identity with the nucleotide sequences of Pakistan’s formerly reported <i>R. microplus</i>, and full-length immunogenic sequences revealed maximum identities to the most similar sequences reported from India, China, Cuba, USA, Brazil, Egypt, Mexico, Israel, and Uruguay. Low nonsynonymous polymorphisms were observed in ATAQ (1.5%), cathepsin-L (0.6%), and aquaporin 2 (0.4%) sequences compared to the homologous sequences from Mexico, India, and the USA, respectively. Based on the <i>cox</i> sequence, <i>R. microplus</i> was phylogenetically assembled in clade C, which includes <i>R. microplus</i> from Pakistan, Myanmar, Malaysia, Thailand, Bangladesh, and India. In the phylogenetic trees, the cystatin 2b, cathepsin-L, ferritin 1, and aquaporin 2 sequences were clustered with the most similar available sequences of <i>R. microplus</i>, P0 with <i>R. microplus</i>, <i>R. sanguineus</i> and <i>R. haemaphysaloides</i>, and GST, ATAQ, and Rm05Uy with <i>R. microplus</i> and <i>R. annulatus</i>. This is the first report on the molecular characterization of clade C <i>R. microplus</i>-derived immunogenic sequences.