If you have a named beneficiary, then that's who the 401(k) money will go to if you pass away.

The law requires that you change your beneficiary to your wife. Right now if something happens your ex-girlfriend would get the 401k money. I am surprised you were able to change the beneficiary for your 401K even though you were still married. Normally you cannot change the beneficiary from your wife unless she gives her consent and signs a form. You should change the beneficiary to someone that is still in your life such as a family member or your wife. Just because you are separated is not a reason not to give her the money you are still married to her legally. If you are planning on getting a divorce then you should do it and then assign the 401K to a family member.

What Reader said is true, but a surviving spouse is mandated by federal law to be the primary beneficiary of a 401k. Did your spouse sign off to waive that right or did you lie on the beneficiary form and claim you were unmarried in order to name someone else?

Oooh, the things I learn on this site! Further research supports IMHO's reply:

Unless your not-quite-ex-wife has agreed and signed off on it, she still has the right to claim your 401(k) money. She may have to go to court to get it, because you've muddied the waters with the girlfriend as beneficiary. But she would win.

The 401(k) isn't the only bit of your estate that might end up in dispute, either. If you truly want to not be married to your wife, get the paperwork done right. Else you will leave more than one unhappy beneficiary.