Management of breeding- and free-living populations, traditionally based on phenotypic traits, relays more and more on availability of reliable information about the basic population genetic parameters as heterozygosity, mean number of alleles per locus, percentage of polymorphic loci, population structuring, genetic distances and others. Therefore, the application of molecular markers, revealing a great deal of phenotypically hidden information, becomes inevitable for population analysis. Conservation geneticists use this information for implementation of appropriate management policies. Application of molecular markers in Lipizzan horse breed, which is an example for a pedigreed breeding population, and in two endangered salmonid fish populations in Slovenia, are presented. In the Lipizzan horse breed, an insight in the population structure, overall heterozygosity, relationship between population parameters and phenotypic traits and reliability of pedigree data was gained by using molecular markers. Marble trout and Adriatic grayling were selected as examples for free-living populations, seriously endangered by human activity in the past. Development of informative molecular markers, their application and suggestions for appropriate conservation actions are described.