Background Lassa virus (LASV) intra- and inter-lineage nucleotide diversity has made it challenging to develop a single-reaction RT-PCR assay that can detect all viral variants with equal sensitivity. We evaluated PANDAA ® LASV, a single-reaction pan-lineage RT-PCR assay, at two Nigerian Lassa fever laboratories against the RealStar ® Lassa Virus RT-PCR Kit 2.0, the current diagnostic standard in Nigeria, which requires two separate reactions per patient sample to mitigate against false negative results. Methodology and Principal Findings We conducted a diagnostic accuracy study using retrospectively ( n = 200) and prospectively ( n = 100) plasma samples that had been collected from patients meeting the standard case definition of suspected Lassa fever for testing by real-time PCR at Abubakar Tafawa Balewa University Teaching Hospital in Bauchi and Federal Medical Centre in Owo. PANDAA LASV ® sensitivity was 98.6% (138/140; 95% CI 94.9–99.8%), specificity 99.4% (155/156; 96.5–100%), and accuracy 99.0% (293/296; 97.1–99.8%). The positive likelihood ratio was 153.8 (21.8–1084.9) and the negative likelihood ratio 0.01 (0.00–0.06). PANDAA Ct values correlated significantly with the reference assay L gene target (Spearman's ρ = 0.80; p < 0.0001) and GPC target (ρ = 0.69; p < 0.0001). Three confirmed discordant results occurred at high Ct values near assay detection limits. Performance was consistent across sites with overlapping confidence intervals for sensitivity and specificity and no difference in median Ct values for any assay target between laboratories. Ct values were significantly lower in patients ≥ 45 years ( p = 0.008). Conclusions and Significance PANDAA ® LASV demonstrated high diagnostic accuracy to detect Lassa virus RNA compared to the dual-reaction reference assay. Reducing the operational burden from two PCR reactions per sample to a single PANDAA ® LASV reaction while maintaining equivalent sensitivity and specificity could have a direct impact on labour and reagent costs, and turnaround time. Clinical validation against additional LASV lineages is an immediate priority.