The champion trainer has dominated the race since its inception in 2008, saddling eight of the first 10 winners, thanks largely to the popular six-times winner Quevega.
Benie Des Dieux was having her first run over hurdles for Mullins, having won each of her three starts over fences since arriving in Ireland, and looked to have plenty on her plate against last year's winner Apple's Jade, who was formerly a Mullins inmate before joining Gordon Elliott.
Apple's Jade looked to be travelling well enough rounding the home turn, but was passed by 33-1 shot Midnight Tour on the run to the final flight and it looked as though a major shock could be in the offing.
However, the Rich Ricci-owned Benie Des Dieux was just finding top gear in behind and finished best up the hill to beat the outsider by half a length.
Mullins said: "We decided to come for this race because there's no mares' chase. What else could she have run in? The Ryanair maybe, but this was the only race worth sending her back over hurdles for.
"After she'd won her first race for us, we did a couple of pieces of work and I texted Rich to say she was like 'Benie Des Douvan' - that was the vibe she was giving us. I tried to keep that under wraps, though!
"When she won last time I told David (Mullins) to not leave Cheltenham behind, win if you can but don't give her a hard race, and that's what he did.
"Ruby was brilliant on her but she only just got home. She really needs a trip and will go way back up in trip and back over fences now.
"She really could be anything, I think a lot of her."
Walsh, who received a two-day ban for using his whip above the permitted level on Benie Des Dieux, said: "We always thought she was a really good mare. She got fractionally outpaced down the hill, but going down the hill I was thinking the others are starting to stop. She stuck her head down and rallied really well."
Ricci, who had seen his hot favourite Getabird beaten in the opening Supreme Novices' Hurdle, said: "You're kicked in the first race and then you're on top of the world in the fifth. It's an amazing sport, fantastic.
"It's so great to have Ruby back. He is different gravy. You saw that there, he's sensational."
Midnight Tour's trainer Alan King said: "She's run a blinder. I had a feeling she would. Coming here I thought she might be running on into third or fourth.
"Turning in I thought she might win, but she's just been run out of it."