Plant genetic engineering has become an invaluable tool in plant research. Although plant transformation is a well-established technique, transgene expression is still unpredictable. Silencing may involve epigenetic modifications or nuclear and chromosomal localization of transgenes. In this way, understanding nuclear structure and organization is important not only for increasing our knowledge of fundamental aspects of the genome but also for taking the greatest advantage of inserting foreign genes and controlling their expression in biotechnological applications. Integrated approaches are clearly required in order to elucidate such complex processes. By combining the analysis of the physical position of transgenes with markers for epigenetic modifications in the plant genome we can better understand the factors affecting transgene expression levels and analyze the genomic environments of differentially expressed transgenes. Medicago truncatula Gaertn. has become a well-known model for the legume family and is used in studies ranging from nodulation to environmental stresses. More recently its use in biotechnology has been explored. In this report we describe the application of fluorescence in situ hybridization (FISH) to detect foreign DNA sequences and to determine the organization of the nucleolar organizer regions (NORs) genes in both metaphase chromosomes and interphase nuclei. We also studied chromatin distribution by immunodetection of epigenetic marks in M. truncatula interphase nuclei from tissue sections. We present evidence that M. truncatula is amenable to this kind of studies, which will in turn contribute to a better exploitation of biotechnology applications for this important plant family.