Sustaining and developing of genetic diversity in winter wheat germplasm is one of the main prerequisite for success in future winter wheat breeding programs. Selection of diverse parents is essential for creation of superior new varieties. Danger of genetic erosion specially exists in smaller breeding programs and selection for limited production area with similar growing conditions. Croatian winter wheat breeding has a long tradition but it is relatively small compared to countries with bigger growing areas. Use of molecular markers in evaluating genetic diversity of winter wheat varieties is limited in Croatian breeding programs. Therefore, aim of this study was to evaluate genetic diversity of Croatian winter wheat varieties using SSR and AFLP markers as a powerful tool for assessing genetic diversity. Forty winter wheat varieties, from three Croatian breeding centres and foreign centres were included in the study. A set of 26 microsatellite primers were used, covering three wheat genomes and 42 chromosomes by 0.66 average genetic distance. The largest calculated distance was between the varieties Zlatna Dolina and Lela (dij=0.98), while most similar varieties were Super Žitarka and Barbara with the distance value of dij=0.21. Four AFLP marker combinations generated 108 polymorphic bands with average of 34 specific bands per primer combination. On the average 27 polymorphic bands were generated with average PIC value of 0.34. Specific polymorphic bands were discriminant for the three varieties Žitarka, Super Žitarka and Barbara, which, therefore, can be used for their identification. Grouping of varieties was in accordance with their origin (breeding centre) and pedigree data.