Li-Fraumeni Syndrome (LFS) is a rare cancer syndrome caused by mutations in the TP53 gene. A number of tumor-associated germline mutations have been described in individuals from various ethnic origins although, thus far, none in affected individuals of German descent. Our work aimed to detect germline mutations in the TP53 gene in five index cases of German and Swiss origin with cancers typical of Li-Fraumeni syndrome. We analyzed all ten coding exons of the TP53 gene by direct sequencing of PCR products. We identified five mutations of which three were found in families with a strong history of LFS in several generations while two likely came about de novo. The five mutations include two missense mutations in exon 4 (Gly105Cys) and exon 7 (Arg248Gln), one deletion of 11 base pairs encompassing the splice acceptor sequence of exon 6 (IVS5-11_-1 del), one nonsense mutation in exon 6 (Arg196Stop), and one duplication of 4 base pairs in exon 8 (1020_1023dupCTGC). The functional consequence of the IVS5-11_-1del mutation on splicing of pre-mRNA was investigated by an in vitro assay. Our study reports the first germline mutations in patients of German and Swiss origin with LFS-related tumors. The frequent identification of de novo germline mutations emphasizes the importance of mutational analyses of the TP53 gene particularly in young patients with malignancies typical for LFS, but without a positive family history of this tumor syndrome.