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Seed shattering is one of the main traits related with the domestication of cultivated rice and with the invasiveness and persistence of weedy rice. Two independent studies in 2006 have indicated that qSH1 in Japonica and Sh4 in Indica rice are major genes governing this trait. However, a wide variation of seed shattering occurs in weedy rice ecotypes from the same geographic region and even within the same ecotype. The aim of this study was to evaluate the nucleotide variability of known and putative genes related to seed shattering in cultivated rice and to identify and validate new genes related to this trait in weedy rice. The qSH1 gene was not associated with seed shattering in the evaluated genotypes. The nucleotide variability of the genes Os01g0849100 and Os08g0512400, previously identified based on a genome-wide resequencing study, was related to seed shattering in rice. The nucleotide variability of three single nucleotide polymorphisms (SNPs) of the OsXTH8 gene, which is related to cell wall biosynthesis, was not associated with seed shattering. However, the high expression of this gene was related to the occurrence of this trait. This study evaluated jointly a series of genes involved in rice seed shattering and indicated that the genes OsXTH8, Os08g0512400 and Os01g0849100 are important for the regulation of this trait in weedy rice in addition to previously described genes. Seed shattering in weedy rice has a more complex regulation than in cultivated rice where few major genes were identified.