The two girls had originally boarded a train in their home village of Balloch, West Dunbartonshire on Friday afternoon, travelling to Glasgow.
They had planned to get a train straight back but a staff member in Glasgow wouldn’t let them back through without tickets.
A spokesperson for ScotRail said that they carried out an initial investigation, which included reviewing CCTV from the station and the train itself.
It is understood that by the time the girls made it past the staff in Glasgow, the last train back to Balloch had already departed.
When reviewing the CCTV footage ScotRail found that the unaccompanied 12-year-old girls boarded the 10.17pm service from Glasgow to Helensburgh Central.
The company said that it followed safety procedures and conducted two separate checks of the train to ensure nobody was onboard before the service terminated.
The girls were not found and the firm says CCTV shows the pair deliberately “hiding underneath the seats” during these checks.
The ScotRail spokesperson said: “An investigation into the incident, which included a review of CCTV, shows that our staff carried out two separate checks of the train and didn’t find the girls on either occasion.
“The safety of our customers is critically important and we will continue to thoroughly examine the footage, and speak to our own staff, to ensure that everything possible is being done to avoid any repeat of this incident.
“We remind all customers that by law only essential journeys should be made at the moment due to lockdown rules.”
The girls had been playing in Balloch and had arranged for a parent to pick them up on the Friday afternoon.
Speaking to the BBC, Bonnie-Louise Greenan, the mother who was supposed to pick them up, said it was the worst night of her life.
She alerted the police when she was unable to find the children, and drove around that evening trying to find them herself.
The pair were discovered by a conductor on Saturday morning after they alerted a passer-by that they were stuck.
The British Transport Police said they were aware of the incident and that they referred it to ScotRail to investigate.