The use of molecular genetic markers (MGMs) has become widespread among evolutionary biologists, and the methods of analysis of genetic data improve rapidly, yet an organized framework in which scientists can work is lacking. Elements of molecular evolution are summarized to explain the origin of variation at the DNA level, its measures, and the relationships linking genetic variability to the biological parameters of the studied organisms. MGM are defined by two components: the DNA region(s) screened, and the technique used to reveal its variation. Criteria of choice belong to three categories: (1) the level of variability, (2) the nature of the information (e.g. dominance vs. codominance, ploidy,…) which must be determined according to the biological question and (3) some practical criteria which mainly depend on the equipment of the laboratory and experience of the scientist. A three-step procedure is proposed for drawing up MGMs suitable to answer given biological questions, and compiled data are organized to guide the choice at each step: (1) choice, determined by the biological question, of the level of variability and of the criteria of the nature of information, (2) choice of the DNA region and (3) choice of the technique.