Coffee is an important crop worldwide, grown on about 10 million hectares in tropical regions including Latin America, Africa, and Asia. The genus Coffea includes more than 100 species; most are diploid, except for C. arabica, which is allotetraploid and autogamous. The genetic diversity of commercial coffee is low, likely due to it being self-pollinating, in addition, the widespread propagation of few selected cultivars, such as Caturra, Bourbon, and Typica. One approach is the analysis of genome size in these cultivars as a proxy to study its genetic variability. In the present work, genome size of 16 cultivars was assessed through high-resolution flow cytometry (FCM). Nuclear DNA was analyzed using a modified procedure that uses propidium iodide (PI) and 4′,6′-diamino-2-phenylindole dihydrochloride hydrate (DAPI) staining. The C. arabica cultivars investigated possessed a nuclear DNA content ranging from 2.56 ± 0.016 pg for Typica, to 3.16 ± 0.033 pg for ICATU, which had the largest genome size. All cultivars measured using both fluorochromes had greater estimates with DAPI than PI. The proportion of the genome composed of guanosine and cytosine (GC%) among the cultivars evaluated in this study ranged from 37.03% to 39.22%. There are few studies of genome size by FCM of distinct important C. arabica cultivars, e.g., hybrids and artificial crosses. Thus, this work could be valuable for coffee breeding programs. The data presented here are intended to expand the genomic understanding of C. arabica and could link nuclear DNA content with evolutionary relationships such as diversification, hybridization and polyploidy.