Abstract The C4 Urochloa spp (syn. Brachiaria ) and Megathyrsus maximus (syn. Panicum maximum ) are used as pasture for cattle across vast areas in tropical agriculture systems in Africa and South America. A key target for variety improvement is forage quality: enhanced digestibility could decrease amount of land required per unit production and enhanced lipid content could decrease methane emissions from cattle. For these traits, loss-of-function (LOF) alleles in known gene targets are predicted to improve them, making a reverse genetics approach of allele mining feasible. We studied allelic diversity of 20 target genes (11 for digestibility, 9 for lipid content) in 104 accessions selected to represent genetic diversity and ploidy levels of U. brizan tha, U. decumbens, U. humidicola, U. ruziziensis and M. maximum . We used RNAseq and then bait-capture DNA-seq to improve gene models in a U. ruziziensis reference genome to assign polymorphisms with high confidence. We found 953 non-synonymous polymorphisms across all genes and accessions; within these, we identified 7 putative LOF alleles with high confidence, including ones in the non-redundant SDP1 and BAHD01 genes present in diploid and tetraploid accessions. These LOF alleles could respectively confer increased lipid content and digestibility if incorporated into a breeding programme. Highlight We found gene variants in a collection of tropical grasses that could help reduce environmental impact of cattle production.