Heavy metals are a group of pollutants in biological sludge. Many agencies regulated guidelines for heavy metal concentrations for various applications of sludge such as agricultural application. In this study, we tried to determine heavy metal fate after anaerobic digestion. Additionally, we determined the bioaccumulation rate of heavy metals in lettuce cultivated on a sludge-applied land. Heavy metal (As, Pb, Hg, Cd) contents of solid and liquid parts of raw and anaerobically digested sludge were separately measured by ICP-OES. For this purpose, the samples were digested using nitric acid, hydrochloric acid, and boric acid. Then, the raw and anaerobically digested sludge were used for cultivation of lettuce in separate farms. The heavy metal concentrations in the harvested lettuce were measured by the same procedure. The results showed that the main part of heavy metals in the raw sludge was in the liquid part (67%), while, the main part of heavy metals in the anaerobically digested samples was in the solid part of the sludge. Because of washout of dissolved heavy metals in the liquid part of the sludge, the lettuce cultivated by anaerobically digested sludge had higher content of the heavy metals in comparison to that of the lettuce cultivated by the raw sludge. This study showed that application of anaerobically digested sludge can increase the bioaccumulation rate of heavy metals in the crops and induce more human health risk.