Axillary bud multiplication and direct adventitious bud regeneration were improved for clonal propagation of Portuguese genotypes of Pinus pinaster. Axillary buds induced in an ex vitro two year-old genotype by 6-benzylaminopurine (BAP) spraying and apex removal were used for the establishment of in vitro axillary bud cultures. Improved procedures were also developed for both axillary bud multiplication from four week-old seedlings and direct adventitious bud induction from cotyledons. The first in vitro multiplication cycle of buds derived from the ex vitro material resulted in an approximately five-fold multiplication rate. Regarding the axillary buds derived from four-week-old seedlings, the best induction medium for bud multiplication was based on a GD medium plus 1.11 µM BAP and 0.27 µM α-naphthaleneacetic acid (NAA). Induction of adventitious buds varied significantly among seed provenances. In the best performing provenance, the highest bud induction frequency (95%) after three multiplication cycles was achieved on a GMD basal medium supplemented with 4.45 µM BAP and 0.05 µM NAA for three weeks. The axillary bud multiplication protocol for seedlings is potentially useful for large-scale propagation of seeds coming from the Portuguese breeding program of P. pinaster.