Molecular markers provide researchers with a powerful tool for variation analysis between plant genomes. They are heritable and widely distributed across the genome and for this reason have many applications in plant taxonomy and genotyping. Over the last decade, molecular marker technology has developed rapidly and is now a crucial component for genetic linkage analysis, trait mapping, diversity analysis, and association studies. This chapter focuses on molecular marker discovery, its application, and future perspectives for plant genotyping through pangenome assemblies. Included are descriptions of automated methods for genome and sequence distance estimation, genome contaminant analysis in sequence reads, genome structural variation, and SNP discovery methods.