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Gillingham’s play-off hopes were ended on Sunday.
A home defeat, coupled with results going against the Gills elsewhere, left them sitting ninth in League 1.
Aiden O’Brien put Millwall ahead early in the second half.
The visitors also hit the crossbar twice, once in each half, through former Gills defender Joe Martin and striker Steve Morison.
John Egan grabbed a late equaliser for the Gills but Lee Gregory fired home from the spot to win it.
Sunday’s near sell-out game at Priestfield was a must-win for the Gills but they were also reliant on both Scunthorpe and Barnsley dropping points. Both teams won, making the result at Priestfield irrelevant.
Gillingham’s form heading into the game was poor, without a win in seven, extended to eight after Sunday’s loss.
Manager Justin Edinburgh made two enforced changes as Arron Morris (knee) and Bradley Garmston (hamstring) dropped out. Replacing them in the XI were skipper Doug Loft and Brennan Dickenson.
Deji Oshilaja remained at right-back as Edinburgh stuck with a 4-4-2 formation for the visit of a Millwall side already guaranteed a play-off place.
The Lions were hoping to keep up their momentum for a possible return to the Championship while also keen to avenge a 3-0 defeat by the Gills at The Den back in December.
It was the visitors who came closest to scoring in the first half. Gills keeper Stuart Nelson had already saved with his feet from O’Brien before denying Mark Beevers with a super save moments later. The follow-up from Martin hit the bar.
Oshilaja had a trio of headers, two chances that went wide and one which was saved by keeper Jordan Archer, and Bradley Dack tried his luck from 25 yards but the low shot was comfortably dealt with.
Oshilaja was close again early in the second half, as he attempted to flick Egan’s header into the net off a Dack cross, but keeper Archer was there to deny the Gills defender.
Gillingham’s hopes nosedived just before the hour mark. Nelson made a great save to deny Morison after the defence had been opened up but O’Brien got onto the loose ball to put Millwall 1-0 ahead.
While that was happening at Priestfield, Barnsley went 3-1 up at Wigan and moments later Scunthorpe added a second to lead Sheffield United 2-0, leaving the Gills well adrift of the coveted sixth place.
A switch in formation to 3-5-2 and a triple substitution was Edinburgh’s last throw of the dice.
Cody McDonald glanced a chance wide for the Gills and Egan also came close, shooting just the wrong side of a post.
Dack also went close, having a shot saved, while Josh Wright fired an effort wide.
Millwall finished with 10 men after former Gills player Mahlon Romeo was shown a straight red card following a rash challenge on Brennan Dickenson. Lions manager Neil Harris was also ordered to the stands after his protests.
Gillingham levelled, with Egan firing home in the second minute of injury-time, but Edinburgh’s men conceded with almost the last kick. Dickenson fouled Gregory in the box and the substitute stepped up to score the winner from 12 yards.
Gillingham: Nelson, Oshilaja, Dickenson, Egan, Ehmer, Loft (Williams 58mins), Hessenthaler (Jackson 58mins), Wright, Dack, McDonald, Samuel (Norris 58mins). Subs not used: G Morris, Donnelly, El-Abd, Osadebe.
Millwall: Archer, Romeo, Martin, Beevers, Webster, Ferguson (Upson 71mins), Taylor, Thompson, Abdou, Morison (Craig 89mins), O’Brien (Gregory 75mins). Subs not used: Forde, Edwards, Williams, Onyedinma.
Attendance: 9,375 (1,213).