Robert Earnshaw fired the Welsh into a first half lead but James Morrison marked his 25th birthday with the equaliser soon after the break, before Kenny Miller and Christophe Berra grabbed a goal apiece.
The last meeting between the two nations resulted in a 3-0 defeat for the Scots in November 2009, which ultimately cost George Burley his job.
But Wednesday night's 3-1 victory means it will be a straight fight between Scotland and the Republic of Ireland to claim the Nations Cup when they meet in the tournament's final game on Sunday.
Ross McCormack earned his first cap since that infamous defeat in Cardiff two years ago and started along with Steven Naismith, as Craig Levein made just two changes from the friendly against Brazil in March.
While the Scots named a strong starting XI, Wales boss Gary Speed left out most of his Premier League players, handing first starts to Neil Taylor and Owain Tudor Jones.
There was also a noticeable difference between the number of fans at the game, with around 2,500 Tartan Army making the trip despite travel disruptions caused by the volcanic ash cloud, compared to just 150 Wales supporters.
It was Scotland who carved out the first real chance when Miller - who was captain in the absence of Darren Fletcher - nodded on for Morrison and he flicked just wide of target.
At the other end, goalkeeper Allan McGregor was forced into action to punch to safety before Sam Vokes could connect with a header from a corner six yards out.
The Scots could have taken the lead when Miller sprayed the ball out to his former Rangers team-mate Naismith who forced a decent block from Boaz Myhill with an angled drive.
Wales started to come into the game more and Jermaine Easter rippled the side netting, before Vokes collected from Andy King and rifled just over from 20 yards.
Scotland squandered a great chance when Steven Whittaker burst down the right flank before squaring for Miller who failed to connect properly and stab home from just a few yards out.
Charlie Adam then saw his shot deflected past the post and Levein's men were left rueing those missed opportunities when Wales surged into the lead after 36 minutes.
Neal Eardley punted a long ball up the park which was missed by Stephen Crainey, allowing Earnshaw to race into the box and loft over McGregor and into the back of the net.
Scotland were back on level terms 10 minutes after the restart when Adam flighted a lovely ball from the halfway line into the path of Morrison and he did well to hold off Christopher Gunter before rifling home.
Miller then passed up the opportunity to fire Scotland into the lead when he fired across the face of goal and wide of the upright, before McCormack sent an overhead kick just over.
But Miller was on target with 64 minutes gone when Morrison turned provider with a simple pass and all that was required from the Bursaspor striker was to slot past Myhill from 10 yards.
Scotland increased their lead with 20 minutes to go when Whittaker's cross picked out Berra at the back post to nod into the back of the net from close range.
Barry Bannan and Barry Robson were thrown into the action and Bannan immediately tested the goalkeeper with a stinging shot but Myhill was up to the task on this occasion.
Norwich's Russell Martin and Grant Hanley of Blackburn both made their international debuts late on as Levein's men held on for the win.