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Bradley Dack’s 15th goal of the season for Gillingham ended Barnsley’s winning run.
Dominic Samuel put the Gills ahead midway through the first half, and though Conor Hourihane levelled after the break, Dack fired the Gills back in front within a minute.
Victory for the Gills ended Barnsley’s dominance of the fixture. The Yorkshire side had won the previous six games between the two sides.
Both teams had proud records to protect ahead of Saturday’s clash.
The Gills boasted the Football League’s best home record and also topped the scoring charts.
Barnsley went into the match with seven successive wins behind them, a run of form which had lifted them from relegation contenders to play-off chasers.
But it was Gillingham’s day on Saturday.
Boss Justin Edinburgh had to make an enforced change to his defence, replacing injured Ryan Jackson with Aaron Morris. Bradley Garmston won a recall to left-back after recovering from his own injury.
Sadly Garmston wouldn’t last the 90, limping off after an hour.
New signing George Williams was named on the bench as Edinburgh kept faith with Rory Donnelly and Samuel in attack.
The two men up front combined to put the Gills ahead with 25 minutes gone, just as the visitors appeared to be getting a stranglehold on the match.
Adam Hammill and Sam Winnall had both gone close for Barnsley before Samuel put the Gills ahead, finishing low past the keeper after Donnelly picked his run out to perfection, capitalising on a loose ball from the visitors.
There wasn’t a great deal separating the sides in the opening 45, with the Gills restricting Barnsley to half-chances. Marley Watkins should have done better with his chance, but his attempted lob lacked height and Stuart Nelson saved easily.
Samuel put an early second-half effort wide for the Gills after being picked out at the near post by Dack. Donnelly put another chance over the bar from the edge of the box.
Barnsley levelled the scores through Hourihane on 62 minutes, the visiting midfielder breaking into the box and beating Nelson with a shot into the far corner.
It took the Gills just 60 seconds to reply. Donnelly’s shot could only be parried by keeper Adam Davies and Dack was on hand to fire home the loose ball.
Substitute Adam Chicksen made a vital clearance, heading the ball out, as Winnall threatened to pounce from a left-sided cross.
Hourihane came close to equalising again in stoppage-time, firing narrowly wide from the edge of the box.
Gillingham: Nelson, A Morris, Garmston (Chicksen 64mins), Egan, Ehmer, Wright, Loft, Osadebe (McGlashan 86mins), Dack, Samuel, Donnelly. Subs not used: Williams, G Morris, Hessenthaler, List, Norris.
Barnsley: Davies, Bree, Long, Mawson, White, Brownhill (Scowen 61mins), Isgrove (Fletcher 61mins), Hourihane, Hammill, Watkins (Khan 86mins), Winnall. Subs not used: Townsend, Roberts, Williams, Tuton.
Attendance: 5,887 (380 away).