Abstract Motivation In the era of multiple genome references, researchers often align sequencing reads against distinct assemblies or even multiple references simultaneously. This enables applications such as the detection of introgressed segments or highly variable genomic regions, which are especially prevalent in large-genome crop species such as lettuce or wheat. However, these applications come at the cost of increased computational burden, inconsistencies in mapping methods, and reduced reproducibility across studies. To address these limitations, we developed KCFtools, a Java-based toolkit that identifies the presence and absence of k-mers in nonoverlapping genomic or transcriptomic windows by comparing query and reference genomes. This alignment-free approach enables the efficient computation of an identity score for each window, thereby facilitating robust detection of introgressed or variable regions across genomes. Results We systematically evaluated the performance and accuracy of the k-mer-based method implemented in KCFtools, benchmarking it against conventional single nucleotide variation-based introgression detection pipelines. Our results demonstrate that KCFtools effectively captures introgressed segments and structurally diverse regions, even in species with fragmented or highly divergent reference genomes. In addition, we extended KCFtools to generate genotype matrices from k-mer variation tables. These matrices are compatible with genome-wide association studies software and allow the identification of loci associated with phenotypic traits. We showcase the utility of this approach by detecting known and novel associations for downy mildew resistance in lettuce, underscoring the pipeline’s potential for high-resolution, reference-agnostic population genetic analysis. Availability and implementation https://github.com/sivasubramanics/kcftools

UNLABELLED: The design of genetic markers is of particular relevance in crop breeding programs. Despite many economically important crops being polyploid organisms, the current primer design tools are tailored for diploid species. Bread wheat, for instance, is a hexaploid comprising of three related genomes and the performance of genetic markers is diminished if the primers are not genome specific. PolyMarker is a pipeline that generates SNP markers by selecting candidate primers for a specified genome using local alignments and standard primer design tools to test the viability of the primers. A command line tool and a web interface are available to the community. AVAILABILITY AND IMPLEMENTATION: PolyMarker is available as a ruby BioGem: bio-polyploid-tools. Web interface: http://polymarker.tgac.ac.uk.