Abstract Recognition of the AUG start codon on mRNAs during translation initiation in eukaryotes occurs in a preinitiation complex that includes small ribosomal subunits and multiple translation initiation factors. The complexity of this process and the lack of appropriate tools have prevented its genetic study in multicellular organisms. Here we describe a genetic system in the nematode Caenorhabditis elegans to study how the AUG start codon is selected. We have generated a sensitive reporter assay that allows for the isolation of mutants with reduced fidelity to recognize the AUG start codon. Two mutants were identified to have dominant missense mutations in iftb-1, which encodes the β-subunit of eIF2 (eIF2β). Both mutations occur in a conserved region located outside of the C2–C2 zinc finger domain where yeast SUI3 mutations are localized in Saccharomyces cerevisiae eIF2β. C. elegans iftb-1, as well as mutant eIF2βs carrying the equivalent SUI3 mutations, are able to initiate translation at non-AUG codons that retain two potential base-pairing interactions with the anticodon of the initiator methionyl tRNA. These analyses further support the critical role of eIF2β in start codon selection, and two functional domains within eIF2β are likely involved, one defined by our C. elegans mutants and the other by the yeast SUI3 mutants.