Evolutive studies have verified that Coffea arabica (2n = 44) is a natural segmental allopolyploid originated from a cross between two diploid (2n = 22) Coffea species. Data obtained by classical cytogenetic analyses showed that C. arabica chromosomes are small and morphologically similar, which hampers the karyogram assembly with well-identified homologue pairs. In the present study, the C. arabica complement was reanalysed using an improved cytogenetic protocol that allowed the obtention of high-quality prometaphasic and metaphasic chromosomes. The results showed that chromosomes are cytogenetically distinct (1, 2, 19, 20, 21 and 22) and identical (3–4, 5–6, 7–8, 9–10, 11–12, 13–14, 15–16 and 17–18), with regard to their total length, short and long arm sizes or chromosome classes. Our work suggests that C. arabica is a true non-segmental allotetraploid but originated from different species exhibiting similar and distinct chromosomes.