Sulfur (S) affects the plant life cycle and crop yield and has nutritional importance for human and animal diet. Its deficiency is one of the major problems in agriculture. However, the plant-specific LSU (response to Low Sulfur) gene family has not been extensively analyzed in major plant species such as grasses. In this study, we have performed in silico genome-wide analysis of LSU genes in 6 grass species, including Brachypodium distachyon, Sorghum bicolor, Oryza sativa, Zea mays, Triticum aestivum, and Panicum virgatum. All identified LSU genes contained one exon encoding proteins of acidic character with cytoplasmic localization. In silico analysis of cis-elements revealed that sulfur-responsive elements (SURE boxes, Sulfur Response Element, GAGAC motif) were present in all LSU genes. In phylogenetic analysis, dicot and monocot LSU genes were separated. Expression profiles of B. Distachyon BdLSU1 and BdLSU2 genes were analyzed by qRT-PCR method. Two Brachypodium LSU genes demonstrated different expression patterns when subjected to 48 h of S-depletion treatment. In roots, the BdLSU2 gene was upregulated, while BdLSU1 was downregulated. In leaves, expression levels were decreased for both genes. Analysis of the BdLSU expression under drought, cold, salt, and heat stresses was carried out based on the Brachypodium stress atlas. Results showed that BdLSU genes are not specific to S limitation; indeed, they may be involved in different stress conditions by cooperating with their interacting partner proteins. The results of this study could significantly contribute to the understanding of LSU genes in plants, particularly in grass species. These results may also support plant molecular studies by aiding the understanding of the sulfur assimilation pathway.