Aguado-Santacruz, G. A., Betancourt-Guerra, D. A., Siquerios-Cendón, T., Arévalo-Gallegos, S., Rivera-Chavira, B. E., Nevarez-Moorillon, G. V., Moreno-Gómez, B. and Rascón-Cruz, Q. 2011. Comparison of the structure and organization of therrnaoperons of Bouteloua gracilisandZea mays. Can. J. Plant Sci. 91: 107-116. We studied the genomic structure of Bouteloua gracilis chloroplast DNA (cpDNA) and compared it with the sequenced ribosomal RNA spacer region from other cereals. This will allow us to understand chloroplast topology and the recombination ability of cpDNA. The development of potential tools for biotechnology applied to cereals can be focused through the study of cpDNA in family related grasses, such as B. gracilis. cpDNA was prepared from green B. gracilis and Zea mays plants using a modified NaCl method. A 2332 bp intergenic spacer (IGS) region (rrna16S-trnI-trnA-rrna23S) from B. gracilis was sequenced, which showed great similarity (at least 92%) to IGS region from Z. mays, Oryza sativa and Saccharum officinarum. A physical map constructed by Southern hybridization using petA, psbA, psbD, ndhA, rbcL, 16S and 23S rDNA digoxigenin-labelled probes showed low organizational resemblance to maize cpDNA. Moreover, when compared to a similar fragment of Z. mays, a 239 bp intron deletion was found in the trnI gene in the B. gracilis cpDNA. Restriction and hybridization analyses suggested that the B. gracilis cpDNA has a molecular weight of 130 Kb. We expect that the findings reported in this work can be a baseline for increasing our knowledge in chloroplast organization in grasses and for the development of molecular tools.