The Reading Eagle newspaper said witnesses reported 50 to 100 dead birds Sunday along Route 222 in Spring Township near Reading.
The Pennsylvania Game Commission launched an investigation and early reports indicate that they were apparently poisoned, legally, by a farmer.
The birds are European starlings, and are considered an invasive species by the United States Department of Agriculture.
Harry Brown of the Animal Rescue League of Berks County said it's unlikely that an illness like West Nile virus would kill that many birds in such a small area. But he said a tractor-trailer could be to blame.
Brown said birds are sometimes caught in the crosswind created by one rig, then struck by another.