Developing improved fruiting varieties of kiwiber- ry (Actinidia arguta) and other species of cold-hardy kiwifruit (e.g., A. kolomikta) is highly resource-inten- sive due to the dioecy, extended juvenile phase (3–5 years), and vegetative vigor of these woody, vining species. The ability to identify gynoecious plants at the seedling stage is therefore desired as a means of increasing the resource-use efficiency of breeding programs. In this study, sex-linked polymorphisms in both species were identified through reference-inde- pendent genotyping-by-sequencing (GBS) analysis; validated in independent populations; and convert- ed into cost-effective, gel-based PCR markers appro- priate for marker-assisted breeding programs. The marker for A. arguta, designed using male-allele-spe- cific primers based on a SNP within a 157 bp GBS am- plicon, exhibits perfect sex-linkage across a compre- hensive set of kiwiberry germplasm currently avail- able through the US nursery trade and the USDA’s National Plant Germplasm System (NPGS). By multi- plexing conserved primers for a co-located internal 81 bp control amplicon, this male-specific marker was made effectively co-dominant, thereby enhanc- ing scoring accuracy. Similarly, the 161 bp marker for A. kolomikta was designed with male-allele-specific primers based on a 3 bp indel within a 270 bp GBS fragment and exhibits perfect sex-linkage throughout the available A. kolomikta germplasm in North Ameri- ca. Both markers were validated in independent pop- ulations and are now being used for high-throughput sex-screening assays of seedling populations.