My Brother died and I just learned that he was still married.  But he has been seperated for over 15 years. Does the wife still have any interest in his estate?

If he was married his spouse still has rights under Mississippi law. If there was a Will naming her she has those rights.  If she renounces the Will even if left nothing in it, she can receive what she would have received as if he died intestate. If no children she receives the entire estate. There needs to be a divorce to eliminate her rights under either his Will if he had one or intestate succession laws if not.