The selection of tolerant varieties is a powerful strategy to ensure highly stable yield under elevated temperatures. In this paper, we report the phenotypic and genotypic characterization of 10 tomato landraces to identify the best performing under high temperatures. The phenotyping of five yield-related traits allowed us to select one genotype that exhibits highly stable yield performances in different environmental conditions. Moreover, a Genotyping-by-Sequencing approach allowed us to explore the genetic variability of the tested genotypes. The high and stable yielding landrace E42 was the most polymorphic one, with ~49% and ~47% private SNPs and InDels, respectively. The effect of 26,113 mutations on proteins’ structure was investigated and it was discovered that 37 had a high impact on the structure of 34 proteins of which some are putatively involved in responses to high temperatures. Additionally, 129 polymorphic sequences aligned against tomato wild species genomes revealed the presence in the genotype E42 of several introgressed regions deriving from S. pimpinellifolium. The position on the tomato map of genes affected by moderate and high impact mutations was also compared with that of known markers/QTLs (Quantitative Trait Loci) associated with reproductive and yield-related traits. The candidate genes/QTLs regulating heat tolerance in the selected landrace E42 could be further investigated to better understand the genetic mechanisms controlling traits for high and stable yield trait under high temperatures.