Detecting latent tuberculosis infection (LTBI) is important, especially in high-risk populations including healthcare workers (HCWs). QuantiFERON-TB Gold Plus (QFT-Plus) is a new version of the interferon-gamma release assays (IGRAs) to replace the QuantiFERON-TB Gold In-tube (QFT-GIT). However, data on the use of QFT-Plus for LTBI detection in high TB-burden countries are limited. This study was conducted in a TB-endemic setting in Thailand. HCWs were enrolled in the study and underwent both tests during the annual health screening. The testing results were compared and the concordance was determined. Of 102 HCWs, 11 (10.78%) were positive according to both tests, and 15 (14.71%) were positive according to QFT-Plus. The overall agreement between assays was 96.08%, with Cohen's kappa coefficient (k) at 0.82. All four discordant results occurred with QFT-GIT negative and QFT-Plus positive. The comparison between QFT-GIT and QFT-Plus based on each antigen tube (TB1 or TB2) exhibited similar concordance with 99.02% and 95.10% agreement, respectively. The intra-comparison between TB1 and TB2 of QFT-Plus also showed good concordance at 96.08%. Among this group of HCWs, the LTBI prevalence of any positive results in both tests was low. Overall, the study showed good agreement between QFT-Plus and QFT-GIT (k = 0.82) with a minimal difference, suggesting similar assay performance to that mainly carried out in TB-low incidence countries. The results support the use of QFT-Plus for detecting LTBI in a format similar to QFT-GIT.