The Upper Bavaria police department said prosecutors in the state capital of Munich had ordered the criminal probe over the crash of the packed regional train on Friday.
The accident occurred near the Alpine resort Garmisch-Partenkirchen, an area gearing up to host the G7 summit in late June.
The investigation is targeting “three (national railway company) Deutsche Bahn staff members on suspicion of offences including criminally negligent homicide”.
Police provided no further details on the case against the suspects.
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Public broadcaster Bayerischer Rundfunk (BR) said investigators probing the cause of the accident were focusing on possible technical defects in the undercarriage of some of the cars, as well as the railway itself.
German media reported at the weekend that Deutsche Bahn had planned maintenance work on the route beginning on June 25th. The company declined to comment.
The crash killed four adult women – two Germans and two Ukrainians – as well as a German teenager aged 14.
More than 40 passengers were hurt, one of whom is still in a critical condition, police said.
The accident occurred just after midday on Friday as school holidays were starting in the two southern German regions Baden-Wuerttemberg and Bavaria.
Police said the regional train was “very crowded” with about 140 people on board as a new €9 monthly public transport ticket valid across Germany also boosted demand.
The train had just left Garmisch-Partenkirchen for Munich when the accident took place in the Burgrain district.
From June 26-28th, heads of state and government including US President Joe Biden are due to meet at Schloss Elmau — 11 kilometres (seven miles) from Garmisch-Partenkirchen.
BR quoted Bavarian interior minister Joachim Herrmann as saying there was “no indication” that the accident could have been caused by an attack on the rails by G7 protesters in the run-up to the summit.
“We can’t rule anything out entirely but based on the investigation so far, nothing points in that direction,” he said.