High seawater temperatures in summer often lead to high mortality in farmed abalone, which greatly increases the cost of farming. The hybrid abalone (Haliotis discus hannai ♀ × H. fulgens ♂, DF), is one of the main varieties of abalone cultured in Fujian Province, China, to resist the high temperature. In this study, the high-temperature resistance of DF abalones was assessed under 28 °C - 32 °C. 31 °C was applied in the subsequent screening experiments of high-temperature resistant individuals. A total of 614,321 single nucleotide polymorphisms (SNPs) were identified via super genotyping-by-sequencing (S-GBS), and 69 SNPs involved in 42 candidate genes were identified by genome-wide association study (GWAS). In addition, according to gene functional annotation, the locus with the lowest P value was screened within the threshold range (−log₁₀ P = 4.98296). The genotyping was verified by Sequenom MassARRAY, and 7 SNPs were proved to be related to high temperature resistance in DF population of 100 individuals. 7 candidate genes (fabp, e3rglg4, rgs2, tanc2x6, osbp1, arsb, and uch1) were identified near these SNPs. Finally, these candidate genes were further proved to be associated with the high-temperature resistance trait of DF by the analysis of differential expression genes between sensitive and tolerant individuals. These candidate genes involved regulatory mechanisms such as cell stability, protein repair, lipid metabolism and immune regulation. Notably, the mechanism by which the e3rglg4 gene was involved in the degradation of abnormally folded proteins of abalone under heat stress, corroborating the reliability of the results of GWAS and Sequenom MassARRA. These results complement the shortage of molecular markers for heat resistance of hybrid abalone, provide a reference for marker-assisted selection (MAS) and accelerate the development of heat-resistant abalone varieties.