Wheat (Triticum aestivum L.), one of the three most important cereal crops worldwide, has a dominant position in Europe due to its adaptability and consumer acceptance particularly as organic food commodity. Organic agriculture is developing rapidly, and its authenticity is presently a subject of great concern to food authorities, as incorrect labeling can represent commercial fraud. A comparative transcriptome profiling was conducted on winter wheat flag leaves of several cultivars growing in open fields under different agricultural production systems. Performing a microarray study, ten transcripts differentially expressed in organic and conventional growing conditions were identified in Tommi and Centenaire cultivars. Transcript abundance profiles of selected probe sets were independently confirmed by quantitative reverse-transcription PCR analysis, tested on Tommi, Centenaire, and Cubus cultivars from different growing year and geographical sites. Univariate and multivariate statistical analysis showed that the global wheat transcriptome is influenced by the agricultural system indicating a promising approach for analytical verification of the production system of wheat at the farm level.