Background: The Significance Analysis of Microarrays (SAM) is a popular method for detecting significantly expressed genes and controlling the false discovery rate (FDR). Recently, it has been reported in the literature that the FDR is not well controlled by SAM. Due to the vast application of SAM in microarray data analysis, it is of great importance to have an extensive evaluation of SAM and its associated R-package (sam2.20). Results: Our study has identified several discrepancies between SAM and sam2.20. One major difference is that SAM and sam2.20 use different methods for estimating FDR. Such discrepancies may cause confusion among the researchers who are using SAM or are developing the SAM-like methods. We have also shown that SAM provides no meaningful estimates of FDR and this problem has been corrected in sam2.20 by using a different formula for estimating FDR. However, we have found that, even with the improvement sam2.20 has made over SAM, sam2.20 may still produce erroneo...