Martyn Woolford and Krystian Pearce netted second-half goals to put Scunthorpe in charge of the first leg tie.
In a narrow first half, both sides cancelled eachother out with few goalscoring chances. Iron midfielder Matt Sparrow was lucky to stay on the field after a late sliding challenge on Millers 'keeper Andy Warrington.
The home side stepped it up after the break as Woolford opened the scoring on the hour beating Warrington from close range after a well-worked move that split open the Millers defence.
Seven minutes later Scunthorpe made it 2-0 when centre-back Krystian Pearce rose at the far post to head home from a corner after a point blank Warrington save moments earlier.
Rotherham nearly hit back late on but Ian Sharps volleyed his shot inches wide after a succession of stoppage time corners and have their work cut out to turn around the defecit in a month's time.
Scunthorpe: Murphy, Byrne, Mirfin, Pearce, Williams, Thompson, Sparrow, Wright, Woolford, Hayes (Morris 90), Hooper (Forte 81).
Subs Not Used: Slocombe, Crosby, Hurst.
Booked: Sparrow, Pearce, Wright.
Subs Not Used: Cann, Joseph, Ryan Taylor.
Booked: Hudson.
Att: 6,038
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