Boots is banning cheque payments at around 1,500 stores in the UK and Ireland from May 1.
The high street chain said usage had dropped to around 0.1% of all payments since chip and pin came in.
It is the latest retailer to restrict cheque book payments in UK stores.
Boots initially trialled the chequebook ban in Surrey and Sussex.
It has issued in-store notices warning customers of the impending nationwide ban.
The retailer said customers had responded "positively" to the chequebook ban during the three month trial in the South East.
Boots spokesman Donal McCabe said: "Since chip and pin has come in the use of cheques has dropped dramatically.
"Unfortunately cheques still pose probably the highest risk of fraud to ourselves and our customers, and because they are increasingly not used we have decided to phase them out completely."
Boots will make some exceptions for customers paying for prescriptions and certain medical goods by cheque.
Petrol giant Shell stopped accepting cheques at its 586 forecourts in the UK in 2005.
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