In recent years, the use of chromogenic media for detection of coliforms and E. coli increases as they allow fast identification of colonies directly on the plate without further confirmation steps. We obtained an atypical strain from a raw milk sample forming bright red colonies on RAPID'E.coli 2 agar, as opposed to the violet coloration characteristic for E. coli, or blue/green coloration characteristic for coliforms. API32E and MALDI-TOF analysis were used to identify isolate 2099 as E. coli. While API ID 32 E analysis resulted in a biochemical profile characteristic for E. coli, isolate 2099 was found to be phenotypically β-glucuronidase positive but β-galactosidase negative, which resulted in atypical red coloration of its colonies on RAPID'E.coli 2 agar. In order to elucidate possible causes of the β-galactosidase negative phenotype, we sequenced the lacZ gene encoding β-galactosidase. A mutation leading to a premature stop codon at amino acid position 774 was identified, rendering the polypeptide abnormally short and most likely not functional. Considering our findings, not only β-glucuronidase, but also β-galactosidase negative E. coli represent a challenge to routine diagnostic procedures screening for E. coli with chromogenic media that rely on detection of β-glucuronidase and β-galactosidase activity.