WV 45 begins at the Virginia state line in the southwestern corner of Berkeley County. The roadway continues across the state line as SR 681 (Glengary Road) in Frederick County, Virginia. WV 45 heads north and then east through a mix of forest and farmland in a valley east of Sleepy Creek Mountain. East of the village of Glengary, the state highway crosses Back Creek then veers northeast to ascend North Mountain. WV 45 intersects WV 51 (Gerrardstown Road) at Mills Gap at the top of the mountain. WV 45 descends into the Shenandoah Valley, passing the community of Union Corner and the historic homes Campbellton and Ar-Qua Springs. In the village of Arden, WV 45 turns east onto Greenhouse Road, south onto Arden Nollville Road, then northeast from the second road to head toward Martinsburg.^[1]

WV 45 intersects I-81 at a diamond interchange and continues east as Apple Harvest Drive, a four-lane undivided highway that passes through a commercial area south of the city limits of Martinsburg where the highway meets a Winchester and Western Railroad line at grade and intersects US 11 (Winchester Avenue). East of the commercial area, WV 45 has a folded diamond interchange with Queen Street, which crosses over the highway. The roadway continues east as WV 9 (Charles Town Road); WV 45 and WV 9 run concurrently north on two-lane Queen Street. The two highways head north through the Boydville Historic District toward downtown Martinsburg, where Queen Street intersects King Street, which carries US 11 west. US 11 joins the two state-numbered highways on Queen Street past the Apollo Theatre and near the Martinsburg station, which serves Amtrak's Capitol Limited and is the western terminus of MARC's Brunswick Line. Queen Street traverses Tuscarora Creek, passes under CSX's Cumberland Subdivision, and passes through the Baltimore and Ohio and Related Industries Historic District.^[1]

WV 45 leaves the concurrency with US 11 and WV 9 at East Moler Avenue, where the highway heads east out of Martinsburg as Martinsburg Parkway, a two-lane road that crosses Opequon Creek and passes through a mix of forest and farmland. The state highway passes through the hamlet of Files Crossroad and enters Jefferson County at Rockymarsh Run just north of Wynkoop Spring. WV 45 passes through the community of Mechlenburg Heights before entering the town of Shepherdstown as German Street. The state highway reaches its eastern terminus at an intersection with WV 480 (Duke Street) in the center of the town. German Street continues east as WV 230, which turns south toward Halltown.^[1]

West Virginia Route 45 Alternate runs southeast from the WV 45 at the east end of Old Martinsburg Road (CR 12/1) along Potomac Farms Drive to West Virginia Route 480 across from the intersection with Morgan Grove Road (CR 16/5) west of Shepherdstown.