Abstract Background: The biomechanical properties of maize stalks largely determine their lodging resistance, which affects crop yield per unit area. However, the quantitative and qualitative relationship between micro-phenotypes and the biomechanics of maize stalks is still under examined. In particular, the roles of the number, geometry, and distribution of vascular bundles of stalks in maize lodging resistance remain unclear. Research on these biomechanical properties will benefit from high-resolution micro-phenotypic image acquisition capabilities, which have been improved by modern X-ray imaging devices such as micro-CT and the development of micro-phenotyping analysis software. Hence, high-throughput image analysis and accurate quantification of anatomical phenotypes of stalks are necessary. Results: We have updated VesselParser version 1.0 to version 2.0 and have improved its performance, accuracy, and computation strategies. Anatomical characteristics of the second and third stalk internodes of the cultivars ‘Jingke968’ and ‘Jingdan38’ were analyzed using VesselParser 2.0. The relationships between lodging resistance and anatomical phenotypes of stalks between the two different maize varieties were investigated. The total area of vascular bundles in the peripheral layer, auxiliary axis diameter, and total area of vascular bundles were revealed to have the highest correlation with mechanical properties, and anatomical phenotypes of maize stalk were better predictors of mechanical properties than macro features observed optically from direct measurement, such as diameter and perimeter. Conclusions: This study demonstrates the utility of VesselParser 2.0 in assessing stalk mechanical properties. The combination of anatomical phenotypes and mechanical behavior research provides unique insights into the problem of stalk lodging, showing that micro phenotypes of vascular bundles are good predictors of maize stalk mechanical properties that may be important indices for the evaluation and identification of the biomechanical properties to improve lodging resistance of future maize varieties.