The aim of this study was to compare the effects of a new organic Se [2-hydroxy-4-methylselenobutanoic acid (HMSeBA)] with routinely used mineral and organic Se sources (sodium selenite and selenized yeast) on chosen performance criteria and Se deposition in egg and muscle of laying hens. A total of 240 laying hens (40 wk of age) were randomly assigned to 6 treatments for 56 d with 8 replicates of 5 hens per replicate. The 6 treatments were as follows: control group received basal diet without Se supplementation; the second, fourth, and sixth experimental groups (SS-0.2, SY-0.2, and HMSeBA-0.2, respectively) were fed basal diet supplemented with Se at 0.2 mg/kg from sodium selenite, selenized yeast, and HMSeBA, respectively; and the third and fifth experimental groups (SY-0.1, and HMSeBA-0.1, respectively) were fed basal diet supplemented with Se at 0.1 mg/kg from selenized yeast and HMSeBA, respectively. No difference was observed among dietary treatments on feed intake, egg weight, and laying rate. All hens fed the Se-supplemented diets exhibited greater total Se contents in their eggs compared with control hens (P < 0.01). The egg Se concentrations were greater in hens fed organic Se (HMSeBA-0.2, P < 0.01, and SY-0.2, P < 0.01) than those fed the SS-0.2. In addition, hens fed the diet with HMSeBA-0.2 accumulated more Se in their eggs (+28.78%; P < 0.01) and muscles (+28%; P < 0.01) than those fed the diet supplemented with SY-0.2. These results showed the greater ability of HMSeBA to increase Se deposition in eggs and breast muscle of laying hens, which can subsequently lead to greater supply of Se for humans.