Aggregative multicellularity relies on cooperation among individual cells to form a multicellular body. InDictyostelium discoideumthis cooperation is maintained by high relatedness. Previous work showed that experimental evolution under low-relatedness resulted in an increase of cheaters (cells that contribute proportionally more to viable spores than to the dead, sterile stalk) and that many clones completely lost cooperation and the ability to form fruiting bodies. Here, we investigate the genetic basis underlying the evolution of the cheating phenotype using whole-genome sequencing and variant analysis of these 24 previously evolvedD. discoideumlines. We identified 38 single nucleotide polymorphisms in 29 genes, none of which have been previously reported to cause cheating and most of which are uncharacterized. Each gene has one unique variant except for the G protein-coupled receptorgrlG, which has at least one variant in over half of the lines. Upon identifying the parallel evolution ofgrlG, we screened additional clones to investigate the correlation between variants in the gene and the complete loss of cooperation (identified by the inability to form a fruiting body). We found that variants in the first half ofgrlGthat impact the signal peptide or extracellular binding domain are significantly associated with the complete loss of cooperation (non-fruiting); the association was not significant in the second half of the gene. Our results suggest that the loss ofgrlGwas adaptive under the experimental conditions of low-relatedness and that the first half of the gene in particular is involved in cooperation and multicellular development inD. discoideum. This represents a new selfish mutation that arose naturally under conditions of low-relatedness and helps confirm the importance of high relatedness in the evolution of altruism in the social amoebaDictyostelium discoideum.