Variation in genome size in a particular taxonomic group can reflect different evolutionary processes including polyploidy, hybridization and natural selection but also neutral evolution. Using flow cytometry, karyology, ITS sequencing and field surveys, the causes of variation in genome size in the ecologically and morphologically diverse high-Andean genus Lasiocephalus (Asteraceae, Senecioneae) were examined. There was a 1.64-fold variation in holoploid genome size (C-values) among 189 samples belonging to 20 taxa. The most distinct was a group of plants with large genomes corresponding to DNA triploids. Disregarding the DNA triploids, the remaining samples exhibited a pronounced (up to 1.32-fold) and rather continuous variation. Plants with the smallest genomes most likely represent intergeneric hybrids with the closely related and sympatric Culcitium nivale, which has a smaller genome than Lasiocephalus. The variation in genome size in samples of diploid Lasiocephalus was strongly correlated with several environmental and life history traits (altitude, habitat and growth form). However, all these factors, as well as genome size itself, were correlated with phylogeny (main split into the so-called ‘forest’ and ‘páramo’ clades), which most probably represents the true cause of the differentiation in intrageneric genome size. In contrast, relationships between genome size and phylogeny were not apparent at lower divergence levels. Instead, here we suggest that ecological conditions have played a role in driving shifts in genome size between closely related species inhabiting different environments. Collectively, this study demonstrates that various evolutionary forces and processes have shaped the variation in genome size and indicates that there is a need for multi-approach analyses when searching for the causes and consequences of changes in genome size.