Molecular markers are essential to dissecting genetic relationships, performing genome-wide association mapping, QTL mapping or genomic selection in the breeding practice. In some organisms like grape, the breeding program includes species that diverged around 20 million years ago, so developing a universal marker panel that work for the whole clade is challenging but crucial. However, in our previous large-scale QTL mapping of different grape populations using genotyping-by-sequencing and AmpSeq platforms, we found that the average success rate of transferring the single nucleotide polymorphisms markers to another species is only 2%. In this study, we developed a core genome-based marker development pipeline for the highly diverse and heterozygous species. In this pipeline, we targeted collinear blocks of the core genome based on 10X Genomics de novo assembled genomes, selected regions of moderate polymorphism from shotgun resequenced samples, and avoided genetic variations under priming sites. Using high-fidelity rhAmpSeq genotyping chemistry, we developed 2,000 rhAmpSeq markers as a PCR multiplex and validated this pipeline in four F1 or F2 populations representing the diversity of the Vitis genus. A total of 91.4% of markers returned data at one-fold depth on average in all populations. For each population, at least 61.6% of markers were placed on the genetic map, and all parental genetic maps were highly correlated  with physical position in the PN40024 reference genome (r = 0.86 to 0.95).  Most importantly, identical markers were associated with flower sex in two populations used for validation, which indicates that not only are random phenotypically neutral markers transferable, but also functional markers are transferable. Combining collinear 10X genomes with high-fidelity rhAmpSeq genotyping platform, our pipeline can provide an efficient, robust, and flexible genotyping platform for molecular breeding and population studies in highly diverse and heterozygous taxa.