Phosphoglucan phosphatases (Like-SEX4 1 and 2; LSF1 and LSF2) were reported to play roles in starch metabolism in leaves of Arabidopsis. In this study, we identified and mapped the LSF1 and LSF2 genes in barley (HvLSF1 and HvLSF2), characterized their gene and protein structures, predicted the cis-elements of their promoters, and analysed their expression patterns. HvLSF1 and HvLSF2 were mapped on the long arm of chromosome 1H (1HL) and 5H (5HL), respectively. Our results revealed varied exon-intron structures and conserved exon-intron junctions in both LSF1 and LSF2 from a range of analysed species. Alignment of protein sequences indicated that cTP and CT domains are much less varied than the functional domains (PDZ, DPS and CBM48). LSF2 was mainly expressed in anthers of barley and rice, and in leaf of Arabidopsis. LSF1 was mainly expressed in endosperm of barley and leaf of Arabidopsis and rice. The expression of LSF1 exhibited a diurnal pattern in rice only and that of LSF2 in both rice and Arabidopsis. Of the investigated stresses, only cold stress significantly reduced expression level of LSF1 and LSF2 in barley and LSF2 in Arabidopsis at late stages of the treatments. While heat treatment significantly decreased expression levels of LSF1 at middle stage (4 h) of a treatment in Arabidopsis only. The strong relationships detected between LSF2 and starch excess4 (SEX4), glucan, water dikinases or phosphoglucan, water dikinases were identified and discussed. Taken together, these results provide information of genetic manipulation of LSF1 and LSF2, especially in monocotyledon and further elucidate their regulatory mechanism in plant development.