The competition will be held from December 1 to 4, 2023, across the Northern and Southern Zones in the country.

Former Champions Hasaacas Ladies will up against Division One League side Blacoe Soccer while Pearlpia Ladies lock horns with Northern Ladies in an all-Tamale affair.

Elsewhere in the Southern Zone, Police Ladies take on Immigration Ladies while Berry Ladies face Jonina Ladies in all Premier League affair for a place in the next round.

Below are the pairings for the Round of 64:
 
Greater Accra Region 
Police Ladies Vs Immigration Ladies 
Berry Ladies Vs Jonina Ladies 
Blessed Ladies VS Ideal Ladies 
FC Epiphany Warriors Vs Anlo Ladies 
Army Ladies Vs GAR Ladies 
Halifax Queens Vs Faith Ladies

Upper East Region 
Zorkor Liberty Ladies vrs Kunkua Ladies FC 

Northern Region 
Yoo Ladies vrs FC Young Queens 
Kumbungu Ladies vrs FC Savannah 
Pearlpia Ladies vrs Northern Ladies 
Bolga All Stars Ladies vrs Tamale Super Ladies 
Anfaani Ladies vrs Nasara Ladies 

Upper West Region
Wa All Stars Ladies vrs Bagabaga Ladies 
Real Crusaders vrs Kpongu Maidens

Brong Ahafo Region 
Fosu Royal Ladies vrs Ampem Darkoa Ladies 
Gyagba Ladies FC vrs Jaman Vision Ladies 
FC Prisons Ladies vrs Ken Hammer Ladies 

Ashanti Region 
Dynamite Ladies draw a bye 
Osei Tutu Ladies vrs Soccer Angel Ladies 
Supreme Ladies vrs Nana Afia Kobi Ampem 
Ashtown Ladies vrs Dreamz Ladies 
Fabulous Ladies vrs Kumasi Sports Academy 

Western Region 
Hasaacas Ladies vrs Blacoe Soccer 
Hassport Ladies vrs Immaculate Ladies 
Wisdom Ladies Fc vrs Leona Ladies

Central Region 
Kessewa Ladies vrs Socrates Maidens 
Soccer Intellectuals vrs Sealions FC 
Essiam Socrates vrs Ali Royal Ladies 
Mfantsiman Royal Ladies vrs Ladystrikers 

Eastern Region 
Valued Girls vrs Kotoku Rush Ladies 

Volta Region 
Rootz Sistaz FC vrs Ketu Ladies 
Agave Glad Ladies vrs Dream Big Ladies