A 100% owner of a company - his wife who has an account balance under the plan died over a year ago, and the owner rolls over his spouse's account balance into a segregated account under his name in the plan - would this be considered an "unrelated" rollover and therefore not counted in the top-heavy test?

Interesting question. Without doing any research to support my opinion, I'd classify this as "related" and therefore must be counted.

My thought on this was that since it is coming from his wife's account and not for example - his account from another plan that is sponsored by the same employer it would be unrelated.

Ok, I decided to quickly look into it a bit. Note that I said quickly - it was by no means a careful analysis of the rules! Take a look at 1.416-1, T-12. I think this will give you your rules on whether or not it would have to be counted. If the deceased would have been counted as key as of the determination date, then it has to be included. (I.E. - did she have 1 year of service in the 1-year period ending on the determination date).