Ralstonia solanacearum is an extremely destructive phytopathogenic bacterium for which there is no effective control method. Though many pathogenic factors have been identified, the survival strategies of R. solanacearum in host plants remain unclear. Transposon insertion sequencing (Tn-seq) is a high-throughput genetic screening technology. This study conducted a Tn-seq analysis using the in planta environment as selective pressure to identify R. solanacearum genes required for survival in tomato plants. One hundred thirty genes were identified as putative genes required for survival in tomato plants. Sixty-three of these genes were classified into four Clusters of Orthologous Groups categories. The absence of genes that encode the outer membrane lipoprotein LolB (RS_RS01965) or the membrane protein RS_RS04475 severely decreased the in planta fitness of R. solanacearum. RS_RS09970 and RS_RS04490 are involved in tryptophan and serine biosynthesis, respectively. Mutants that lack RS_RS09970 or RS_RS04490 did not cause any wilt symptoms in susceptible tomato plants. These results confirmed the importance of genes related to "cell wall/membrane/envelope biogenesis" and "amino acid transport and metabolism" for survival in plants. The gene encoding NADH-quinone oxidoreductase subunit B (RS_RS10340) is one of the 13 identified genes involved in "energy production and conversion," and the Clp protease gene (RS_RS08645) is one of the 11 identified genes assigned to "posttranslational modification, protein turnover, and chaperones." Both genes were confirmed to be required for survival in plants. In conclusion, this study globally identified and validated R. solanacearum genes required for survival in tomato plants and provided essential information for a more complete view of the pathogenic mechanism of R. solanacearum. IMPORTANCE Tomato plant xylem is a nutritionally limiting and dynamically changing habitat. Studies on how R. solanacearum survives in this hostile environment are important for our full understanding of the pathogenic mechanism of this bacterium. Though many omics approaches have been employed to study in planta survival strategies, the direct genome-wide identification of R. solanacearum genes required for survival in plants is still lacking. This study performed a Tn-seq analysis in R. solanacearum and revealed that genes in the categories "cell wall/membrane/envelope biogenesis," "amino acid transport and metabolism," "energy production and conversion," "posttranslational modification, protein turnover, chaperones" and others play important roles in the survival of R. solanacearum in tomato plants.