Background and Aims Nicotiana section Suaveolentes comprises similar to 65 allotetraploid species derived from South American diploid ancestors. This group originated around six million years ago, with diversification in arid central Australia, coinciding with successive chromosomal reorganizations forming a descending dysploid series (n = 24-15). However, genome size variation does not parallel chromosome number variation, possibly due to changes in repetitive DNA. To clarify this non-parallel evolution, we investigated the relationship between karyotypic diversification, repetitive DNA variability and constitutive heterochromatin distribution.Methods We performed comparative analyses of repetitive DNA by clustering sequence reads with RepeatExplorer, chromosome banding with CMA/DAPI, and FISH using telomeric, ribosomal and satellite DNA probes.Key Results Our results revealed extensive heterochromatin variation, mainly terminally located, with Ty3/Gypsy LTR retrotransposons associated with larger genomes, whereas satellite DNAs were more abundant in species with fewer chromosomes. Terminal CMA+ heterochromatin linked to Tsate181 satellite DNA predominated in species with lower chromosome numbers, whereas those with higher numbers showed greater transposable element content. The number and position of 5S rDNA loci varied during diversification, reflecting descending dysploidy, whereas those of 35S rDNA remained largely constant.Conclusions The contrasting dynamics of retrotransposons, accumulated in larger genomes, and satellite DNAs, amplified in species with lower chromosome numbers, suggest that genomic turnover might have contributed to adaptation to arid environments and facilitated colonization of novel habitats.