Reciprocal hemizygosity analysis is a genetic technique that allows phenotypic determination of the allelic effects of a gene in a genetically uniform background. Expanding this single gene technique to generate a genome-wide collection is termed as reciprocal hemizygosity scanning (RHS). The RHS collection should circumvent the need for linkage mapping and provide the power to identify all possible allelic variants for a given phenotype. However, the published RHS collections based on the existing genome-wide haploid deletion library reported a high rate of false positives. In this study, we report de novo construction of a RHS collection that is not based on the yeast deletion library. This collection has been constructed for the shortest yeast chromosome, ChrI. Using this ChrI RHS collection, we identified 13 allelic variants for the previously mapped loci and novel allelic variants for the growth differences in different environments. A few of these novel variants, which were fine mapped to a gene level, identified novel genetic variation for the previously studied environmental conditions. The availability of a genome-wide RHS collection would thus help us uncover a comprehensive list of allelic variants and better our understanding of the molecular pathways modulating a quantitative trait. © 2014 John Wiley & Sons, Ltd.