Methods involving gel-permeation chromatography used to determine the molecular weight of walnut peptides were studied by comparing two commonly used gel columns. These included a TSK-GEL G2000 SWXL 7.8 mm × 300 mm column, recommended by the national standard GB/T 22492-2008 Soy Peptide Powder and GB/T 22729-2008 Oligopeptide Powder of Marine Fish, and a Superdex Peptide HR 10/30 column, recommended by the industry standard SB/T 10634-2011 Freshwater Fish Collagen Peptide Powder by the Ministry of Commerce. Data were measured, analyzed, and compared for four pure standards and four walnut peptide samples with different molecular weights in each of the two chromatographic conditions. The results showed that for TSK- GEL G2000 SWXL 7.8 mm × 300 mm column, the separation of walnut peptides was achieved within 30 min under the recommended conditions. The results also showed a good linear relationship, with a correlation coefficient of 0.97 and a clear peak shape. The measured molecular weights were higher than the actual values in the regions of >10,000 u and <1,000 u. For the Superdex Peptide HR 10/30 column in comparison, the separation was achieved after 80 min, however the results showed a better linear relationship, with a correlation coefficient of 0.99; however, spilt peaks and broad peaks appeared in the chromatogram. Moreover, in this system, the measured molecular weights were lower than the actual values in the regions of >10,000 u and <1,000 u.