Summary: In medieval central Europe, rye was one of the most important agricultural crops. We combined archaeobotanical methods and ancient DNA sequencing of historical rye material to study patterns of genetic diversity across four centuries. We applied archaeobotanical methods to characterize rye material acquired from construction material ranging from the 14th to 18th centuries from different locations in Germany. Next, we extracted DNA to sequence complete chloroplast genomes of six individual samples and compared sequences of historical rye samples to chloroplast genomes of other cereal crops, including a modern rye cultivar. Comparing the aDNA chloroplast samples with modern and non-domesticated rye chloroplast, we show that genetic variation in the historical German rye population was considerably higher. The exceptional difference in levels of genetic variation likely reflects the consequences of late domestication and selective breeding on genetic variation in this important crop in the last few centuries.