Nine additional marker chromosomes in children with mental retardation and congenital malformation were investigated by routine cytogenetic and in situ hybridization techniques. Five metacentric non-satellited markers and four satellited markers of unknown origin were determined by routine and banding staining. To determine the origin of small marker chromosomes a special scheme involving the sequential application of definite alphoid and "classical" satellite DNA probes with the relative chromosome specificity was employed. The probes specific to four groups of definite chromosomes (i) 1, 3, 5, 6, 7, 10, 12, 16, 19; (ii) 2, 4, 8, 9, 13, 14, 15, 18, 20, 21, 22; (iii) 1, 11, 17, X; (iiii) 9, 13, 14, 15, 21, 22, Y and in situ hybridization under low stringency conditions were used at the first stage of experiments. After the preliminary analysis and the determination of possible origin of a marker chromosome from a definite group of chromosomes the probes with a strong chromosome-specificity under high stringency conditions were used. The approach involving the application of the original collection of chromosome-specific DNA probes, including molecular markers to practically all human chromosomes [1, 2], and various conditions of hybridization provides an effective method for detecting unknown markers. Marker chromosomes investigated in this study were derivatives of chromosomes 7, 9 (two cases), 13, 14, 21 (two cases), X and Y.