<p>In <i>Saccharomyces cerevisiae</i>, the AUG codon typically signals translation initiation to set an open reading frame, with a preference for the A^− 3AA/U^− 1(AUG)U^+ 4 sequence context. The mutant eIF5^G31R or eIF2β^S264Y proteins cause translation initiation fidelity defect by initiating translation at a near cognate UUG codon, in addition to the AUG start codon (suppressor of initiation codon; Sui¯ phenotype). However, the critical role of the − 3 to − 1 sequences in selecting the UUG start codon by these Sui¯ mutants is not fully explored. Different <i>HIS4</i>^<i>UUG</i><i>-LacZ</i> reporter constructs were made with UUG as the start codon and varied nucleotides at the − 3 and − 2 positions or individually at -1 positions. These constructs were transformed to yeast cells having eIF5^G31R or eIF2β^S264Y mutation, and the β-galactosidase activity was measured. The <i>HIS4</i>^<i>UUG</i><i>-LacZ</i> transcripts carrying a purine (A/G) at the − 3 position showed higher reporter activity than those with a pyrimidine (U/C). Additionally, purines were favored at the − 1 position within an AA (− 3 and − 2) context for efficient UUG start codon selection. Our findings demonstrate that UUG start codon recognition by Sui¯ mutants eIF5^G31R and eIF2β^S264Y is greatly influenced by the surrounding nucleotide context, each showing distinct context preferences. This highlights the nuanced role of sequence context in the near-cognate UUG codon initiation by the Sui¯ mutants.</p> Graphical Abstract <p></p>