Flax (
Linum usitatissimum
) is a cool season crop commercially cultivated for seed oil and stem fibre production. A comprehensive characterization of the
heat shock factor
(
HSF
) candidate genes in flax can accelerate genetic improvement and adaptive breeding for high temperature stress tolerance. We report the genome-wide identification of 34 putative
HSF
genes from the flax genome, which we mapped on 14 of the 15 chromosomes. Through comparative homology analysis, we classified these genes into three broad groups, and sub-groups. The arrangement of HSF-specific protein motifs, DNA-binding domain (DBD) and hydrophobic heptad repeat (HR-A/B), and exon-intron boundaries substantiated the phylogenetic separation of these genes. Orthologous relationships and evolutionary analysis revealed that the co-evolution of the
LusHSF
genes was due to recent genome duplication events. Digital and RT-qPCR analyses provided significant evidence of the differential expression of the
LusHSF
genes in various tissues, at various developmental stages, and in response to high-temperature stress. The co-localization of diverse cis-acting elements in the promoters of the
LusHSF
genes further emphasized their regulatory roles in the abiotic stress response. We further confirmed DNA-binding sites on the LusHSF proteins and designed guide RNA sequences for gene editing with minimal off-target effects. These results will hasten functional investigations of
LusHSFs
or assist in devising genome engineering strategies to develop high-temperature stress tolerant flax cultivars.