Abstract The aim of this paper is to elucidate relationship between anatomical changes and lignin deposition dynamics in the cell wall of barley (Hordeum vulgare L.) internodes during four growth stages: heading, flowering, grain filling and ripening. Microscopy was used to analyze anatomical changes related to lignin deposition whereas peroxidase activity was spectrophotometrically determined. In transversal sections lignin was found to be predominant in the sclerenchyma ring in cortex, where particularly lignified cell walls were found. Peroxidase activity was increased in older internodes and their localization in situ was in positive correlation with tissue lignification. Our results showed that, depending on the cultivar, at the flowering and grain filling stages intensive lignin synthesis and deposition occurred. This showed that deposition of lignin in the cell wall at a particular growth stage is in correlation with the lodging resistant phenotype of the investigated cultivars. The results contribute to the understanding of the lignin deposition process during barley development and consequential cell wall thickness.