Bacterial wilt, incited by Ralstonia solanacearum, is a major disease affecting pepper (Capsicum annuum) production worldwide. The most effective management tactic is the deployment of wilt-resistant varieties. However, the lack of a nondestructive method to measure invasiveness and spatio-temporal distribution of R. solanacearum, a vascular pathogen, in planta limits better understanding of pepper resistance and plant-pathogen interactions. We evaluated the resistance of 100 pepper lines using R. solanacearum strain Rs-SY1 (phylotype I, isolated from a sweet pepper in South China). Based on the disease severity index (DSI) values, the elite inbred line BVRC 1 and the small-fruited accessions PI 640435 and PI 640444 were identified as resistant (DSI: 1.2, 1.8, and 1.9 out of 4.0, respectively). In order to evaluate bacterial infection dynamics in planta in real time, we generated seven bioluminescent R. solanacearum strains (BL-Rs1 to BL-Rs7) using vector pXX3 carrying luxCDABE genes, and selected BL-Rs7 for inoculation due to its similarity with parent strain Rs-SY1 in morphology, pathogenicity, and highest light emission in vitro. Luminescence intensity was strongly correlated to bacterial population in planta (R2 = 0.88). The utility of the bioluminescence assay was validated by comparing R. solanacearum infection dynamics in real-time in vivo between resistant line BVRC 1 and susceptible line BVRC 25. The distribution and multiplication of BL-Rs7 strain in resistant line BVRC 1 was conspicuously limited in plants inoculated in either roots or stem compared with susceptible line BVRC 25. These results suggest that pepper line BVRC 1 may resist colonization by interfering with R. solanacearum multiplication in the roots and stem.