Insertion/deletion events (indels) and nucleotide substitutions at the extreme 3′ end of the chloroplast gene matK have been identified that distinguish certain major lineages of grasses. A 1‐bp (base pair) deletion creating a shift in the open reading frame (ORF) and a point mutation support the positions of Streptochaeta and Anomochloa as the two most basal lineages in Poaceae. Another 1‐bp deletion resulting in early termination of the ORF is unique to Ehrharta, a member of the taxonomically disputable tribe Ehrharteae. A 6‐bp insertion supports monophyly of subfamilies Panicoideae, Arundinoideae, Centothecoideae, and Chloridoideae (PACC). This marker appears useful in defining PACC clade members and may have potential in providing insight into the sister‐group relationship between PACC and other lineages. Alignment of deduced amino acid sequences from bryophytes, gymnosperms, and angiosperms shows that this region is relatively conserved, but variation is notably higher in Poaceae. The evolutionary implications of these changes in grasses and other plant families are addressed.