Using the whole-genome data of lettuce, 35 LsHSF genes were identified from lettuce through bioinformatics methods, and named LsHSF1, LsHSF2, …, LsHSF35 according to the distribution order on their chromosome. Their physicochemical properties, chromosomal localization, evolutionary relationship with Arabidopsis, protein conserved motifs, tissue expression levels, and expression levels under high-temperature conditions after melatonin treatment were further analyzed in this study. The results showed that subcellular localization prediction analysis indicated that, except for LsHSF11 and LsHSF17 located in chloroplasts, and LsHSF34 located outside the vacuole, the rest of the genes were located in the nucleus. The 35 LsHSF genes were unevenly distributed on 9 chromosomes. Protein conserved motif prediction analysis revealed that all LsHSF genes contained two closely related Motif 1 and Motif 2. From phylogenetic analysis, HSF proteins were divided into three groups: A, B, and C and could be further subdivided into 14 major subfamilies. Tissue expression level analysis results indicated that HSF genes exhibited specific expression patterns. High-temperature treatment could stimulate the expression of HSF gene family members.