Alstroemeria (Liliales) has two layers of petaloid tepals, in which the often spotted narrow inner tepals can be distinguished easily from the wider outer tepals. In order to explore this floral morphology in Alstroemeria, we investigated the tepal morphology and the expression patterns of three class B genes, whose homologs in eudicots have been shown previously to be involved in petal and stamen development. The two DEF-like genes (AlsDEFa and AlsDEFb) and the one GLO-like gene (AlsGLO) of Alstroemeria ligtu were isolated by rapid amplification of cDNA ends (RACE). Northern hybridization, reverse transcription–PCR (RT–PCR) and in situ hybridization analyses indicated that AlsDEFb and AlsGLO were expressed in whorls 1, 2 and 3 (outer tepals, inner tepals and stamens, respectively), whereas AlsDEFa expression was detected only in whorls 2 and 3. These results suggest that in A. ligtu, AlsDEFb and AlsGLO would participate in determining the organ identity of the two-layered petaloid tepals and stamens, which is in support of the modified ABC model. Additionally, the distinctive expression patterns of AlsDEFa and AlsDEFb might be related to morphological differences between the two-layered tepals.