Genetic modifications of the lignin pathway in alfalfa (M. sativa) were studied by two pyrolysis methods and subsequent multivariate data analysis. Two key genes, hydroxycinnamoyl transferase (HCT) and p-coumourate 3-hydroxylase (C3H), in the lignin biosynthesis pathway were downregulated by the expression of anti-sense RNA. Both genes are involved in the initial two steps in the biosynthetic pathways leading to guaiacyl (G) and syringyl (S) lignin but are not involved in production of p-hydroxyphenyl (H) lignin. Pyrolysis-Molecular Beam Mass Spectrometry (py-MBMS) and pyrolysis-Gas Chromatography Mass Spectroscopy (py-GCMS) were used to investigate the impact of changes in total lignin content and ratios of lignin monomers. The resulting mass spectra from the respective mutants were further investigated by multivariate statistical analysis, such as principal component analysis and hierarchical clustering. We found that down regulation of HCT and C3H resulted in reduced S- and G- lignin and an increased the proportion of H lignin. This is consistent with the respective functions these genes in the lignin biosynthesis pathway. We conclude that py-MBMS is a rapid method for estimating lignin content and the ratios of the monolignols. The effect of the genetic modifications on individual components measured in the pyrolysis vapors can easily be seen and the differences quantified with the multivariate analysis.