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Global warming accelerates the development of high temperature (HT)- and high humidity (HH)-tolerant varieties. This is further facilitated by the identification of HTHH-tolerant genes and the development of molecular markers based on these genes. To identify genes involved in HTHH tolerance in cabbage (<i>Brassica oleracea</i> var. <i>capitata</i>), we performed RNA-seq analysis of two inbred lines, BN1 (HTHH-tolerant) and BN2 (HTHH-susceptible), and selected trehalose 6- phosphate phosphatase I-2 (<i>BoTPPI-2</i>) as one of the HTHH-tolerant-associated genes. We also developed a segregating F₂ population from a cross between BN1 and BN2. RNA-seq results showed that <i>BoTPPI-2</i> transcript levels were high in the HTHH-tolerant inbred line BN1, but not detectable in the HTHH-susceptible inbred line BN2. The expression pattern of <i>BoTPPI-2</i> was not related to the expression of heat shock-related genes. Soft rot resistance, used as an indicator of HTHH tolerance, was higher in BN1 than in BN2. F₂ individuals similar to BN1 with respect to phenotype appeared to be HTHH-tolerant, whereas BN2-types were susceptible to HTHH. Analysis of the genomic DNA revealed the presence of a long terminal repeat (LTR; ca. 4.6 kb) in the ninth intron of the <i>BoTPPI-2_BN2</i> allele, thereby suppressing its transcription and exhibiting HTHH phenotype. Except for the LTR insertion, the sequence of <i>BoTPPI-2_BN2</i> was almost identical to that of <i>BoTPPI-2_BN1</i>. On the basis of the LTR and <i>BoTPPI-2</i> sequences, we developed a molecular marker to identify HTHH-tolerant genotypes and validated its efficiency using F₂ individuals, inbred lines, and cultivars from diverse sources. The marker explained the genetic basis of HTHH tolerance in at least 80%, but not 100%, of the cabbage genotypes. Thus, additional markers associated with HTHH tolerance are needed for perfect selection.