Reliable and accurate laboratory assays to detect recent HIV-1 infection have potential as simple and practical methods of estimating HIV-1 incidence in cross-sectional surveys. This study describes validation of the limiting-antigen (LAg) Avidity enzyme immunoassay (EIA) in a cross-sectional national survey, conducted in Swaziland, comparing it to prospective follow up incidence. As part of the Swaziland HIV-1 Incidence Measurement Survey (SHIMS), 18,172 individuals underwent counselling and HIV rapid testing in a household-based, population survey conducted from December 2010 to June 2011. Plasma samples from HIV-positive persons were classified as recent infections using an incidence testing algorithm with LAg-Avidity EIA (ODn <1.5) followed by viral load (VL ≥1,000 copies/mL). All HIV-seronegative samples were tested for acute HIV-1 infection by nucleic acid amplification test (NAAT) pooling. HIV-seronegative individuals who consented to follow-up, were retested approximately 6 months later to detect observed HIV-1 seroconversion. HIV-1 incidence estimates based on LAg+VL and NAAT were calculated using assay-specific parameters and were compared with prospective incidence estimate. A total of 5,803 (31.9%) of 18,172 survey participants tested HIV-seropositive; of these 5683 (97.9%) were further tested with LAg+VL algorithm. The weighted annualized incidence from the longitudinal cohort study was 2.4% [95% CI 2.0, 2.7]. Based on cross-sectional testing of HIV-positives with LAg+VL algorithm, overall weighted annualized HIV-1 incidence was 2.5% [2.0, 3.0], while NAAT-based incidence was of 2.6%. In addition, LAg-based incidence in men (1.8%; 1.2-2.5) and women (3.2%; 2.4-3.9) were similar to estimates based on observed incidence (men=1.7%, women=3.1%). Changes in HIV-1 incidence with age in men and women further validate plausibility of the algorithm. These results demonstrate that the LAg EIA, in a serial algorithm with VL, is a cost-effective tool to estimate HIV-1 incidence in cross-sectional surveys.