Rovers looked set to make it four points on their travels to Essex this week having beaten Colchester 1-0 on Tuesday night.
But Betsy came off the bench on 84 minutes and slotted the ball home from close range following Dougie Freedman's centre from the left byline.
The Shrimpers had the perfect start on three minutes when Simon Francis' cross from the right was cleared into the path of Theo Robinson.
The on-loan Watford striker saw his initial shot parried by veteran goalkeeper John Achterberg, making his first start since August after Danny Coyne was injured at Colchester, but he composed himself to net at the second attempt from 12 yards.
But Ian Moore levelled for the play-off pushing visitors from the penalty spot after Freedman handled a Chris Shuker cross into the box.
Moore fired his spot-kick low into the right-hand corner of the goal beyond Steve Mildenhall's dive on 62 minutes.
Earlier Francis fired a 30-yard free-kick straight at Achterberg, while the visitors' first attempt on target came midway through the half when Chris Shuker fired in a right-foot effort from 30 yards which was saved by a diving Mildenhall.
Both sides struggled to create much after that until three minutes before the break when a Francis corner from the right fell to Peter Clarke but Achterberg blocked his shot with his legs.
The 38-year-old stopper then turned a rising Robinson effort out for a corner as the half ended.
Tranmere had the first say of the second-half when Charlie Barnett fired straight at Mildenhall from just inside the box.
Southend replied with a flowing move involving Freedman and Robinson, the latter set-up Alan McCormack for an effort which Achterberg blocked with his legs.
Tranmere then got their equaliser and looked the brighter of the two sides, although they failed to test Mildenhall, with only Shuker blasting a shot straight at the stopper.
At the other end of the pitch Freedman had an effort from close range ruled out for offside before Betsy grabbed the late winner.