The systematic position and generic differentiation of the morphologically and geographically outstanding tribe Epithemateae (Gesneriaceae) was analyzed using the rbcL/atpB-spacer and trnL-F intron-spacer regions of chloroplast DNA. In our analysis Epithemateae forms a strongly supported monophyletic clade (bootstrap [BS] = 100%; jackknife [JK] = 100%; decay index [DI] = 12) and appears as sister to the rest of the paleotropical Gesneriaceae (= subfamily Cyrtandroideae). The paleotropical Gesneriaceae form a monophyletic group (BS = 88%; JK = 85%; DI = 3) that is sister to the neotropical Gesneriaceae (subfamily Gesnerioideae) plus Austral Gesneriaceae (subfamily Coronantheroideae) (BS = 99%; JK = 98%; DI = 10). Within Epithemateae Rhynchoglossum is sister to the remaining Epithemateae (BS = 97%; JK = 96%; DI = 12), in which Epithema is sister to a clade of two genera: Loxonia/Stauranthera (BS = 68%; JK = 64%; DI = 1), which form, together with Epithema, a sister clade (BS = 85%; JK = 83%; DI = 2) to Whytockia and Monophyllaea. While the support for Loxonia and Stauranthera is moderate, the relationship of Whytockia and Monophyllaea is very strongly supported (BS = 100%; JK = 100%; DI = 13). Apart from the somewhat surprising (but well-substantiated) isolated position of Rhynchoglossum, the results are in perfect accordance with the relationships worked out earlier on grounds of architectural and floral characters. Especially remarkable is the predicted coherence between the morphologically and geographically different genera Whytockia and Monophyllaea.