Supplementation of wheat bread with maize, rye and oat flours provides higher amounts of protein, dietary fibres, antioxidants, minerals and vitamins. Minimum levels of non-wheat flours in composite breads are regulated by legislation. Thus, objective methods are needed to detect their supplementation levels in bread. In this study, convenient SYBR green-based real time PCR assays using genome specific primers were developed for species identification and quantification in wheat-based composite breads. Three PCR assays targeting gliadin, pML1, secalin and avenin genes for wheat, maize, rye and oat were validated by considerations of a single laboratory procedure. The cereal flour contents in bread with 20% maize, 30% rye and 15% oat flours were quantified as 21.46% +/- 3.90%, 34.43% +/- 7.12% and 12.54% +/- 3.59%, respectively. The limit of detection and limit of quantification values of the assays were 0.086%, 0.410% for maize, 0.166%, 0.808% for rye and 0.119%, 0.290% for oat in wheat-based composite bread. These assays were able to identify and quantify cereal flours in bread successfully and have potential as rapid and sensitive methods for routine detection and quantification of adulteration in wheat-based composite breads. This approach is also applicable to the analysis of all bakery products including any variety of wheat, maize, rye, oat.