Study was conducted to determine the concentrations of (Ni) and (Cr) in sediment and to investigate the effect pH and organic matters on concentrations of nickel and chromium in sediment at Juru River, Penang, Malaysia. The sequential extraction procedure was used based on defined fractions: exchangeable, acid reduction, oxidation and residual. The concentrations of Ni and Cr in residual fraction were higher than the other fractions. In non-residual fractions, the concentration of heavy metals in organic matter fraction was much higher than other fractions (6.63 ± 3.90 μg/g^-1 dry weight ) for nickel and (6.44 ± 2.10 μg/g^-1 dry weight) for chromium. The pH of the sediment in all sites was acidic. The mean of total concentrations of Ni and Cr was 38.21 and 24.92 μg/g^-1 dry weight, respectively. Results of organic matter analysis showed that the percentage of organic matter present in sediment samples varied throughout the river and all sites of sediments were ranged from 7.10% to 16.20%. Comparison between sampling sites using one-way analysis of variance showed that there were significant differences for Ni (P < 0.05) while there was no significant differences for Cr (P > 0.05) in changeable fraction. At acid reduction, oxidation and residual fractions, there were significant differences for the two metals (P < 0.05). For anthropogenic metals, Ni was varied significantly (P < 0.05) whereas Cr was not found significantly (P > 0.05). Analysis of pH and percentage of organic matter present in sediment conclude that Ni and Cr were not strongly correlated at oxidation fraction.