Resistance to acetolactate synthase (ALS) inhibiting herbicides in annual ryegrass has evolved widely in Saudi Arabia but mutations conferring resistance have never been reported. In survey of 91 fields, representing seven ecological regions in Saudi Arabia four (57%) populations had resistance; whereas two (29%) populations had tendency of resistance development against ALS-inhibition herbicides. Use of ALS-inhibiting herbicides provided up to 73% control of ryegrass. Ten base pair substitution evolved in public sphere by the alignment of ALS cDNA sequence. Phenotypic alteration found in five mutants subsequently induced changes in amino acid. The Pro-197-Thr and Pro-197-Gln in Tabouk and Harad population, respectively present at earlier reported location with novel changes in amino acid threonine was unreported. Novel ALS-allele represented by Lys-98-Met mutants had methionine amino acid observed in whole Saudi population except resistant one with lysine. From Asp-185-Tyr novel allele was identified, aspartic acid in all Saudi population as well as in resistant allele and transformed into tyrosine. There exists resistance in ryegrass against ALS-inhibiting herbicides, which suggests use of alternate herbicides and/or development of integrated weed management strategies for wheat in Saudi Arabia.