The game will see Tranmere maintain their annual trip to Saltergate following Chesterfield's relegation to Coca-Cola League Two last season and is a fixture in which Tranmere have had great success in recent years.
Elsewhere in the draw, local side Vauxhall Motors today failed to beat Kidderminster Harriers, meaning the Motormen have not made it to the First Round as Kidderminster go on to play Ware away from home.
Other interesting ties will see Altrincham take on Millwall, Hereford hosting Leeds United, Mansfield at home to either Grays or Lewis, Hartlepool making the trip to Gainsborough, Morecambe hosting Port Vale, Torquay at home to Yeovil Town and Bury vs. Workington