Modeling, simulation, and thermal performance analysis of a thermocycler for the continuous-flow polymerase chain reaction (CF-PCR), with a phase changing material (PCM)-laden annealing flow path, is presented. The incessant threat of microorganisms such as viruses, bacteria, and fungi has fostered effective, quick, and miniature detection devices in order to curtail the wide-spreading of infections. Microfluidics-based CF-PCR systems are compact and ideal for faster response. The thermal cycling process involves a sequential exposure of a given liquid sample to various temperature conditions when it is taken through the continuous-flow path. As a result, a prescribed periodic change of temperature suitable for deoxyribonucleic acid (DNA) amplification is achieved. A rapid temperature reduction and maintenance of isothermal conditions to facilitate the annealing phase of CF-PCR process by a PCM-assisted cooling is envisaged in the present study. Unsteady, two-dimensional, incompressible fluid flow, and internal convection heat transfer in a microchannel annealing path with melting of tetracosane (C24H50) boundary has been simulated using semi-implicit method for pressure linked equations-consistent (SIMPLEC) algorithm based finite volume solver. Solver validation is carried out against the experimental data on internal convection heat transfer in a rectangular microchannel. A detailed numerical study has been performed to assess the spatiotemporal heat transfer characteristics of internal convection in the microfluidic path when the flow triggers the melting of encapsulated PCM. A minimum sample flowrate with PCM encapsulation of less than 600 μm is found to be ideal for achieving desired thermal performance. The present study evidences the swift temperature reduction and management of isothermal conditions congenial for the annealing process in the CF-PCR system for various sample flowrates and PCM masses. The study offers valuable design input for the development of a microfluidic thermocycler for CF-PCR applications.