This paper presents a karyological and cytogenetic characterisation of Artemisia absinthium var. absinthium and A. absinthium var. calcigena (Asteraceae, Anthemideae). Genome size (assessed as nuclear DNA amount), chromosome number, karyotype parameters, fluorochrome banding and fluorescent in situ hybridisation (FISH) of 18S–5.8S–26S and 5S rDNA were studied in Polish specimens of both varieties whose phylogenetic relationships were also established based on their molecular structure. The present study is the first with a focus on A. absinthium var. calcigena. The results basically agree with those previously reported from other A. absinthium populations and show no essential genome organization differences between the varieties.