We applied multicolor spectral karyotyping (SKY) to a panel of 29 newly diagnosed pediatric pre B-cell ALLs with normal and abnormal G-banded karyotypes to identify cryptic translocations and define complex chromosomal rearrangements. By this method, it was possible to define all add chromosomes in six cases, a cryptic t (12; 21)(p13; q11) translocation in six cases, marker chromosomes in two cases and refine the misidentified aberrations by G-banding in two cases. In addition, we identified five novel non-recurrent translocations–t (2; 9)(p11. 2; p13), t (2; 22)(p11. 2; q11. 2), t (6; 8)(p12; p11), t (12; 14)(p13; q32) and t (X; 8)(p22. 3; q?). Of these translocations, t (2; 9), t (2; 22) and t (12; 14) were identified by G-banding analysis and confirmed by SKY. We characterized at (12; 14)(p13; q32) translocation by FISH, and identified a fusion of TEL with IGH for the first time in ALL. We identified a rearrangement of PAX5 locus in a case with t (2; 9)(p11. 2; p13) by FISH and defined the breakpoint telomeric to PAX5 in der (9) t (3; 9)(?; p13). These studies demonstrate the utility of using SKY in combination with G-banding and FISH to augment the precision with which chromosomal aberrations may be identified in tumor cells.