Abstract: SDD1 is a crucial gene regulating stomatal development and encodes a subtilisin-like serine protease. Screening an ethyl methane sulfonate (EMS)-mutagenized population of Arabidopsis thaliana, we isolated two mutants (e281 and g204) with altered stomatal characteristics. The two mutants showed about a 1.5-fold increase in stomatal density and index and formed a clustered stomata similar to the sdd1-1 mutant, but the internal leaf architecture was not altered. Genetic analysis and sequencing results confirmed that e281 and g204 are two novel SDD1 alleles. The mutation results in amino acid changes in the N domain for the substrate binding site and in one of the S domains for the catalytic triad, respectively. Moreover, morphological and physiological data indicate that mutation of different sites within the SDD1 gene lead to different phenotypes. The antagonism among SDD1 alleles can be a negative effect of the gene transvection.