In the last decade, we characterised Portuguese populations of Pinus sylvestris L., Pinus nigra Arnold and Pinus pinaster Aiton by using different molecular and cytogenetic approaches. DNA markers helped assess intra- and inter-population genetic variability, extrapolation of phylogenies, provenances and/or infraspecific taxonomy. Quantitative real-time polymerase chain reaction (qRT-PCR), Classical Cytogenetics techniques and/or Comet assay were valuable to assign origins, infraspecific taxa or populations more tolerant to water stress. Seed germination tests coupled with the cytogenetic analysis of root cell division provided clues about the impact of fire recurrence on the natural regeneration ability and root growth, respectively. Molecular Cytogenetics contributed to detecting chromosomal anomalies commonly detected in individuals from peripheral populations relative to the species’ natural distribution area. The studies briefly described in this chapter integrated multidisciplinary R&D projects whose assembled results allowed inferences about the adaptive potential of the analysed Pinus spp. and relevant information for the definition of strategies concerning germplasm conservation, management, use, and, ultimately, genetic improvement.