Coffea arabica L. is highly homozygotic and caffeine content in most cultivars is reported to be similar (approximately 1%). The caffeine content was analysed using individual seeds of cultivars Catuaí Vermelho, Mundo Novo and Bourbon Vermelho, and varieties Mokka and Laurina of C. arabica. Seeds were cut transversely in half, a thin slice from each was extracted in a mixture of chloroform and methanol, and caffeine was measured by high performance liquid chromatography. The extraction procedure was validated by comparison with two conventional methods less suited for analysing large numbers of seeds. The results revealed an unexpectedly large variation [Catuaí Vermelho: 3.72–25.9 mg g− 1 (seeds); Mundo Novo: 5.06–18.59 mg g− 1; Bourbon Vermelho: 6.76–16.59 mg g− 1] even in types naturally low in caffeine such as Laurina (1.17–9.97 mg g− 1) and Mokka (2.01–9.64 mg g− 1). The mean values for all cultivars were always in the range of the caffeine content found in the literature. Several studies showed that C. arabica has approximately 10% cross-pollination, but the variation observed in caffeine content in individual seeds was too large to be explained by cross-pollination.