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Background: Development of an evaluation tool to determine genotypic variation in phosphorus (P) utilization efficiency is essential to ensure crop productivity and farmers’ income under low P environments. Aims: This study aimed to develop an evaluation tool to determine genotypic variation in low-P tolerance and P use efficiency under low P environments. Methods: Root response and P efficiency traits in 20 maize genotypes with contrasting root systems were assessed 32 days after transplanting into the semi-hydroponic root phenotyping system under low P (10 μM) or optimal P (200 μM) supply. Results: Compared to optimal P, low P supply increased root-to-shoot biomass ratio by 48.7% (shoot dry weight decreased by 20.0% and root dry weight increased by 20.6%). Low P supply increased total root length by 17.8% but decreased primary root depth, with no significant change in lateral root number across all genotypes. Low P stress enhanced P utilization efficiency. Based on genotypic variation and correlations among the 17 measured plant traits in response to low P stress, nine traits were converted to low-P tolerance coefficients (LPTC), compressed by principal component analysis. The three principal component scores were extracted for hierarchical cluster analysis and classified the 20 genotypes into three groups with different P efficiency, including two P-efficient genotypes and nine P-inefficient genotypes. Conclusions: The study demonstrated genotypic variation in response to low P stress. The P-efficient genotypes with higher LPTC values better adapted to low P environments by adjusting root architecture and re-distributing P and biomass in plant organs. The systematic cluster analysis using selected traits and their LPTC values can be used as an evaluation tool in assessing P efficiency among the genotypes.