Sorghum bicolor is a drought- and heat-tolerant cereal widely cultivated in arid regions. However, like other important grasses, it is susceptible to ergot disease caused by Claviceps species, which infect ovary tissues and reduce grain yield. To combat this disease, targeted biotechnological strategies are needed. In this study, we conducted a genome-wide identification and classification of lipid transfer proteins (LTPs) in S. bicolor, uncovering 68 putative genes grouped into five families. In silico analyses of gene expression and promoter sequences identified two LTP genes, SbLTP1.4 and SbLTP1.5, as strong candidates for flower-specific biotechnological applications. Their promoters were functionally tested in transgenic Arabidopsis thaliana, revealing distinct spatial and stress-responsive expression profiles. The SbLTP1.4 promoter was active in stigmas, floral peduncles, styles, and young leaves, and was strongly induced by both biotic and abiotic stress. In contrast, SbLTP1.5 promoter displayed low and stigma-specific expression, with no detectable stress responsiveness. Promoter analysis also revealed conserved transcription factor binding sites across orthologous genes in other economically important grasses, supporting the potential transferability of this strategy to related species. These findings introduce novel tissue-specific promoters for the targeted expression of antimicrobial proteins in reproductive tissues, offering a promising tool to control Claviceps infections and enhance crop resilience in cereals and forage grasses.