<p>Hemp (<i>Cannabis sativa</i> L.) is a versatile crop with substantial potential for creating productive, sustainable, and resilient agricultural systems. However, in contrast to other crops such as cereals, hemp is highly heterozygous, resulting in both challenges and opportunities for agriculture, breeding, and research. Here, we utilise the heterozygosity of hemp to explore the genetic basis of phenotypic variability in a population generated from a single self-pollinated hemp plant. The S1 population shows extensive variability in plant growth, development, and reproductive patterns. Using reduced representation sequencing, selection of alleles heterozygous in the parent plant, and a model originally developed for genome-wide association studies (GWAS), we were able to identify statistically significant single nucleotide variants (SNVs) and haplotypes associated with phenotypic traits of interest, such as flowering time or biomass yield. This new approach, which we term genome-specific association study (GSAS), enables the mapping of traits in a single generation without the need for a large number of diverse cultivars or samples. GSAS might be applicable to other highly heterozygous vegetable and fruit crops, informing the breeding of new cultivars with enhanced uniformity and improved performance in traits relevant to various applications.</p>