Shiga toxin-producing Escherichia coli (STEC) have led to outbreaks worldwide and are considered emerging pathogens. Infections by STEC in humans have been reported after consumption of mainly beef, but also deer. This study investigated the occurrence of STEC in deer in Germany. The virulence genes eae, e-hlyA and saa, the stx subtypes, pulsed-field gel electrophoresis (PFGE) patterns and serovars were studied. In total, 120 samples of 60 animals were screened by real-time polymerase chain reaction (PCR). The PCR results showed a high detection rate of stx genes (83%). Mainly faecal samples, but also some lymphatic tissue samples, tested stx-positive. All isolates carried stx2, were eae-negative and carried e-hlyA in 38% and saa in 9% of samples. Serovars (O88:[H8], O174:[H8], O146:H28) associated with human diseases were also identified. In some animals, isolates from lymphatic tissue and faecal samples showed undistinguishable PFGE patterns. The examined deer were shown to be relevant reservoirs of STEC with subtype stx2b predominating.