This follows the derailment of the operator’s freight train in East Palestine near the Ohio-Pennsylvania border.
The derailment caused a massive fire and released toxic vinyl chloride contained in the rail cars, prompting evacuation of nearby residents.
In a preliminary report, NTSB linked the incident to an overheated wheel bearing.
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Recently, Norfolk Southern vowed to add around 200 detectors to its tracks to detect overheated bearings.
Last week, another Norfolk Southern train derailed in Springfield, Ohio, though no toxic materials were involved this time.
As a result of multiple recent Norfolk Southern accidents, the agency recommended the operator to implement immediate steps to assess its safety measures.
The agency urged the company to take the support of staff and others to make the required changes.
NTSB announced investigation teams for five significant accidents linked to Norfolk Southern, since December 2021.
These accidents include the two recent derailments along with the death of a Norfolk Southern conductor in Cleveland after being hit by a dump truck on 7 March.
NTSB will also review the Norfolk Southern derailment incident in Sandusky, which happened on 8 October last year, as part of the special investigation.
In response to the spate of accidents, Norfolk Southern President and CEO Alan H. Shaw has pledged to improve its safety practices.
Shaw stated: “Moving forward, we are going to rebuild our safety culture from the ground up. We are going to invest more in safety. This is not who we are, it is not acceptable, and it will not continue.”