Aerial parts of 49 species, distributed in 39 genera, all previously included in Scrophulariaceae, but now distributed in several families, have been examined for the presence of betaines. Glycinebetaine was detected in all the plants, but the yield was always low, varying from 0.001% (based on dry weight) from Rhodochitonsanguineum to 0.2% from Rhinanthus minor. Trigonelline was also found in small amounts in Leucophyllum texanum and Chelone oblique, but not in any of the other species. No significant qualitative nor quantitative difference was detected between the betaines found in the examined species of Scrophulariaceae (sensu stricta) and the species tested that were previously included in that family, but are now located in other families, such as Veronicaceae, Orobanchaceae and Calceolariaceae. All these should be considered as families that do not accumulate betaines.