Molecular markers are essential to dissecting genetic relationship, performing genome-wide association mapping, QTL mapping or genomic selection in the breeding practice. In some organism like grape, the breeding program includes species that diverged several millions years ago, therefore, developing a universal marker panel that work for the whole clade is crucial. In our previous large-scale QTL mapping in different grape populations using genotyping-by-sequencing and Ampseq platforms, we found that the markers that worked for one population were not transferable to the other populations in grape. In this study, we developed a pan-genome based marker development pipeline for the highly diverse and heterozygous species. In this pipeline, we considered the collinearity of the core-genome, the polymorphism level of the target regions, and also avoided genetic variations at the probes. Using the high-fidelity rhAmpSeq genotyping platform, we developed 2000 rhAmpSeq markers and validated this pipeline in four F1 or F2 populations of the Vitis genus. A total of 91.4% of markers returns data at one fold depth on average in all populations. For each population, at least 61.6% of markers are placed on the genetic map and the parental genetic maps are highly consistent across all population.  Most importantly, the markers that associated with the flower sex in two population are the same, which indicate that not only random markers are transferable, but also the functional markers are transferable. Combined with the high-fidelity rhAmpSeq genotyping platform, our pipeline can provide an efficient, robust, and flexible genotyping platform for molecular breeding and population study in highly diverse and heterozygous species or the whole genus.