To find and ascertain phenotypic differences, minimal variation between biological replicates is always desired. Variation between the replicates can originate from genetic transformation but also from environmental effects in the greenhouse. Design of experiments (DoE) has been used in field trials for many years and proven its value but is underused within functional genomics including greenhouse experiments. We propose a strategy to estimate the effect of environmental factors with the ultimate goal of minimizing variation between biological replicates, based on DoE. DoE can be analyzed in many ways. We present a graphical solution together with solutions based on classical statistics as well as the newly developed OPLS methodology.In this study, we used DoE to evaluate the influence of plant specific factors (plant size, shoot type, plant quality, amount of fertilizer) and rotation of plant positions on height and section area of 135 cloned wild type poplar trees grown in the greenhouse. Statistical analysis revealed that plant position was the main contributor to variability among biological replicates and applying a plant rotation scheme could reduce this variation.