The foliar rust caused by Melampsora larici-populina (Mlp) is the main disease affecting poplar plantations in Europe. The biotrophic status of this fungus is a major limitation to address in planta transcripts profiling. Thus, identification of reference rust genes steadily expressed during plant tissue colonization is a crucial point. A quantitative PCR approach to assess fungal ITS amplification profile and Reverse Transcription quantitative-PCR was set to compare candidate reference genes amplification profiles in poplar infected tissues. We selected two M. larici-populina genes encoding an alpha-tubulin and the elongation factor-1-alpha that showed the highest expression stability across biological samples and for which transcript levels were correlated with fungal ITS amplification during time-course infection of poplar leaves. We report the use of these reference genes to assess in planta expression profiles of two genes involved in thiamine biosynthesis (THI1 and THI2) for which specific haustorium expression was previously described in the bean rust fungus Uromyces fabae. Mlp-THI1 and Mlp-THI2 showed similar expression profiles. Trancripts were barely detectable in urediniospores as well as during the early stages of infection compared to those reported in the bean rust, whereas a strong induction was observed after haustorial formation after 24 hpi. These data are in frame with the results obtained in U. fabae and consistent with a metabolic reorientation that likely occurs after the fungus derived nutrients from its host in the haustorial structure essential for fungal biotrophy.