Human Immunodeficiency Virus (HIV) diagnosis remains the gateway to HIV care and treatment. However, due to changes in HIV prevalence and testing coverage across different geopolitical zones, it is crucial to evaluate the national HIV testing algorithm as false positivity due to low prevalence could be detrimental to both the client and the service delivery. Therefore, we evaluated the performance of the national HIV rapid testing algorithm using specimens collected from multiple HIV testing services (HTS) sites and compared the results from different HIV prevalence levels across the six geopolitical zones of Nigeria. The evaluation employed a dual approach, retrospective, and prospective. The retrospective evaluation focused on a desktop review of program data (n = 492,880) collated from patients attending routine HTS from six geopolitical zones of Nigeria between January 2017 and December 2019. The prospective component utilized samples (n = 2,895) collected from the field at the HTS and tested using the current national serial HIV rapid testing algorithm. These samples were transported to the National Reference Laboratory (NRL), Abuja, and were re-tested using the national HIV rapid testing algorithm and HIV-1/2 supplementary assays (Geenius to confirm positives and resolve discordance and multiplex assay). The retrospective component of the study revealed that the overall proportion of HIV positives, based on the selected areas, was 5.7% (28,319/492,880) within the study period, and the discordant rate between tests 1 and 2 was 1.1%. The prospective component of the study indicated no significant differences between the test performed at the field using the national HIV rapid testing algorithm and the re-testing performed at the NRL. The comparison between the test performed at the field using the national HIV rapid testing algorithm and Geenius HIV-1/2 supplementary assay showed an agreement rate of 95.2%, while that of the NRL was 99.3%. In addition, the comparison of the field results with HIV multiplex assay indicated a sensitivity of 96.6%, the specificity of 98.2%, PPV of 97.0%, and Kappa Statistic of 0.95, and that of the NRL with HIV multiplex assay was 99.2%, 99.4%, 99.0%, and 0.99, respectively. Results show that the Nigeria national serial HIV rapid testing algorithm performed very well across the target settings. However, the algorithm’s performance in the field was lower than the performance outcomes under a controlled environment in the NRL. There is a need to target testers in the field for routine continuous quality improvement implementation, including refresher trainings as necessary.