The issue that played the GREATEST role in increasing tension and conflict between northern and southern states before the Civil War was:
C. westward expansion and the issue of whether slavery would be allowed in the new territories.
The debate over the extension of slavery into new territories acquired during westward expansion, such as those resulting from the Mexican-American War, led to significant conflict, including the passage of the Kansas-Nebraska Act and violent confrontations known as "Bleeding Kansas." This issue became a central point of contention between the North and South, ultimately contributing to the outbreak of the Civil War.