In comparative phylogenetic and population genetic studies, one of the most crucial steps is to select appropriate DNA markers, a decision based primarily on the estimated variation in markers in cross-taxonomic surveys. To assess whether genetic variation at the intraspecific level in one species predicts the variation in another closely related species we used two congeneric species of Cereus (Cactaceae: Cereeae). We screened and characterized eight noncoding plastid regions (trnS-trnG, atpI-atpH, trnT-trnL, psbD-trnT, petL-psbE, 3'rps16-5'trnk, trnG intron, and trnL intron), and one nuclear gene (PhyC) in Cereus fernambucensis and C. hildmannianus. A total of 40 individuals from 15 populations were characterized according to nucleotide diversity, number of haplotypes, and number of potentially informative characters. The results revealed that nucleotide substitutions and indels are the main source of variation, with the largest divergence between species found in trnS-trnG. The trnL intron and petL-psbE showed intraspecific variability in both species. The psbD-trnT, atpI-atpH, trnS-trnG, and trnT-trnL, which are the most variable regions in one species, showed no variation in the other. Finally, the nuclear gene PhyC showed more resolution between Cereus species than within species. We thus found considerable heterogeneity among widely used plastid markers, even between closely related species, and suggest the use of PhyC as a marker for phylogenetic inference in these species. These results reinforce the need of screening as a preliminary step to conduct phylogeographic or phylogenetic studies in face of unpredictable sequence variation of molecular markers in plants.