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In small plot experiments conducted in maize, the mostly used pollination methods are open and self-pollination treatments. Comparative studies using these treatments are abundant in scientific literature; however studies on the effect of cross pollination and kernel weight on kernel biochemical properties in different treatments are limited. In this study, we conducted a comparative experiment to investigate the effect of pollen contamination and mean kernel weight on kernel biochemical composition of ten different maize genotypes by using two different pollination methods.Open and self-pollination treatments were applied as pollination methods. Eight different traits; kernel weight, cross pollination rate, protein, oil, carbohydrate, oleic acid linoleic acid and carotenoid content were measured. Regression analyses were performed to understand the effects of cross pollination and mean kernel weight on biochemical constituents of maize kernel in different treatments. Results showed that the major biochemical traits, such as protein, oil and carbohydrate content were significantly affected by pollen contamination but minor traits were not. When data were combined (n=60) the effect of pollen contamination in different treatments was not clearly understood. When data (n=30) of each treatment were separately analyzed, it was found that cross pollination rate had significant effect on the most of biochemical constituents in open pollination. Overall, results suggested that pollen contamination had an effect on major biochemical traits in maize and hand pollination could be used for preventing of unwanted effect of pollen contamination in small plot experiments. However, it should be considered that the effects of hand pollination on kernel weight affect the some biochemical traits in maize.