All the big railroads are unionized, per the Railway Labor Act of c.1926.
Short lines often aren't unionized. Some of the "regionals" are, others aren't.
I don't think the Florida East Coast is union -- they had a very bitter strike in the early 1960's that drove the unions out. They may still be "out" to this day.
In summary... if you're going to run the trains, you're probably gonna have to join the union.
Having said that, the operating jobs on the big RR's aren't like they once were. It used to be a much better place to work. Not so much any more. Truth is, the railroad unions have never been noted for "being on the strong side"...