Recently, we used the 5'-trnL(UAA)-trnF(GAA) region of the chloroplast DNA for phylogeographic reconstructions and phylogenetic analysis among the genera Arabidopsis, Boechera, Rorippa, Nasturtium, and Cardamine. Despite the fact that extensive gene duplications are rare among the chloroplast genome of higher plants, within these taxa the anticodon domain of the trnF(GAA) gene exhibit extensive gene duplications with one to eight tandemly repeated copies in close 5' proximity of the functional gene. Interestingly, even in Arabidopsis thaliana we found six putative pseudogenic copies of the functional trnF gene within the 5'-intergenic trnL-trnF spacer. A reexamination of trnL(UAA)-trnF(GAA) regions from numerous published phylogenetic studies among halimolobine, cardaminoid, and other cruciferous taxa revealed not only extensive trnF gene duplications but also favor the hypothesis about a single origin of trnF pseudogene formation during evolution of the Brassicaceae family 16-21 MYA. Conserved sequence motifs from this tandemly repeated region are codistributed nonrandomly throughout the plastome, and we found some similarities with a DNA sequence duplication in the rps7 gene and its adjacent spacer. Our results demonstrate the potential evolutionary dynamics of a plastidic region generally regarded as highly conserved and probably cotranscribed and, as shown here for several genera among cruciferous plants, greatly characterized by parallel gains and losses of duplicated trnF copies.