Despite a prolongation of patient survival, the overall response of doxorubicin (DX) treatment on patients with hepatocellular carcinoma (HCC) remains modest. This is largely attributed to the development of tumor drug resistance either at the onset or during the course of treatment. To investigate the genetic changes associated with DX chemo-resistance, we examined the cytotoxic effect of DX on a panel of 9 HCC cell lines (HepG2, Hep3B, PLC/PRF/5, and six in-house established, HKCI-1, 2, 3 and 4, C1 and C2). The karyotypic abnormalities were examined by spectral karyotyping (SKY) and the chromosome loci defined were investigated for underlying deregulated genes by positional expression profiling. Quantitative RT-PCR was employed to verify the profiling findings, and also used to examine a number of drug resistance-related candidate genes (MDR1, MRP1, MGMT, PTEN, BCL2, BAX, TP53 and P21). Our results indicated that the cytotoxic effect of DX in cell lines exhibited IC50 values that ranged from sensitive to resistant (0.07 to 3.55 microM). While the overall chromosome aneuploidy did not correlate with DX resistance, aberrations on chromosome 10 demonstrated significant correlation with increasing IC50 (p=0.007). Positional profiling further suggested the consistent down-regulation of CGI-18 and ECHS1 on chromosome 10q. The array findings were substantiated by quantitative RT-PCR, which further pointed to a repressed ECHS1 expression in correlation with DX resistance (p=0.021). Among the candidate genes studied, an inverse relationship of P21 (p=0.034) and BAX (p=0.002) expression with DX resistance was also indicated. Our present study highlights the usefulness of multimodality approaches in identifying genetic markers, and further describes the novel finding of ECHS1 down-regulation in the DX chemo-resistance of HCC.