Abstract Vancomycin-resistant enterococci (VRE) can rapidly spread through hospitals. Therefore, our hospital employs a screening program whereby rectal swabs are screened for the presence of vanA and vanB , and only PCR-positive broths are cultured on VRE selection agar. Early November 2016, a clinical vanA- / vanB -negative VRE isolate was detected in a vanA/vanB -screening-negative patient, giving the possibility that an undetected VRE might be spreading within our hospital. Whole-genome-sequencing of the isolate showed that resistance was vanD -mediated and core genome multilocus sequence typing showed it was a rare type: ST17/CT154. To determine the prevalence of vanA/B/C/D -carrying enterococci, we designed a real-time PCR for vanC1/2/3 and vanD and screened rectal swabs from 360 patients. vanD was found in 27.8% of the patients, yet culture demonstrated only E . faecium from vanA -positive broths and E . gallinarum from vanC1 -positive broths. No vanD -positive VRE were found, limiting the possibility of nosocomial spread of this VRE. Moreover, the high prevalence of non-VRE vanD in rectal swabs makes it unfeasible to include the vanD PCR in our VRE screening. However, having validated the vanC1/2/3 and vanD PCRs allows us to rapidly check future vanA/B -negative VRE for the presence of vanC and vanD genes.