Self-incompatibility is a genetic mechanism to prevent self-fertilization and the consequent inbreeding and fitness decay in plant populations. In the gametophyte self-incompatibility mechanism represented by Solanaceae, Rosaceae and Plantaginaceae, the self/non-self recognition between pollen and pistil is determined by the polymorphic S locus. SLF (S-locus F-box)/SFB (S-haplotype-specific F-box) gene is the pollen S determinant of gametophyte self-incompatibility. In this study, 21 SLF/SFB family genes were screened and identified based on the genome-wide of the pineapple. Each AcSLF gene had an F-box conserved domain at the N-terminal. There were great differences in amino acid length, theoretical isoelectric point and molecular weight. The size range of the encoded amino acid sequence was 242-612 aa, and the molecular weight was 27.778-69.070 kDa. The isoelectric point ranged from 5.70 to 9.57. 18 AcSLF proteins were alkaline and most proteins were unstability, only AcSLF19, AcSLF21 were stability. It was predicted that 18 AcSLF proteins were located in the nucleus, one AcSLF protein in chloroplast, two AcSLF protein in both chloroplast and nucleus, and more than 60% of the secondary structure of the protein was composed of alpha helix and random coil. Bioinformatics analysis showed that the pineapple SLF/SFB family genes, which were unevenly distributed on 14 chromosomes, and the chromosome location of AcSLF21 was undetermined. Furthermore, AcSLF4, AcSLF5, AcSLF6, AcSLF8, AcSLF14 and AcSLF15 were linked with RNase T2 family genes, this was one of the main characteristics of pollen SLF gene. AcSLF genes contained different numbers of introns, most of which contained 1~2 introns, and AcSLF3 contained 4 introns. Based on the above analysis results, it showed that AcSLF proteins had different characteristics. Phylogenetic analysis indicated that the evolutionary relationship of AcSLFs in pineapple was far away from the SLF/SFB in Rosaceae, Plantaginaceae and Solanaceae, the degree of clustering was low on the branches of evolutionary tree. The expression levels of the AcSLF genes in pistil, stamen, leaf, sarcocarp, stem and root were analyzed by qRT-PCR. The results showed that AcSLFs had obvious expression differences in different tissues of pineapple. AcSLF1, AcSLF2, AcSLF4, AcSLF9, AcSLF10, AcSLF11, AcSLF15, AcSLF19, AcSLF20 and AcSLF21 genes were highly expressed in pineapple stamens. The expression of AcSLF3 and AcSLF7 in sarcocarp was higher than that of other tissue, and AcSLF5, AcSLF12, AcSLF18 were higher expression in root than other tissues. In general, most pineapple AcSLF genes are mainly expressed in pineapple stamens or leaves. It was found that the expression of AcSLF1, AcSLF2, AcSLF15 genes in pollinated pistil were significantly up-regulated than that in non-pollinated pistil. Especially at 6 h after cross-pollination, the expression level was significantly higher than that of non-pollinated pistils. With the extension of the pollen tube in the pistil, the expression level decreased, but it still had a high expression at 24 h after pollination. And the expression trend of AcSLF1, AcSLF2 and AcSLF15 in cross-pollination was consistent with the growth of pollen tube in the pistil. Therefore, it is speculated that AcSLF1, AcSLF2 and AcSLF15 play an important role in pineapple self-incompatibility. This is the first time to analyze the expression pattern of SLF/SFB family genes in pineapple. This study would provide a reference for the cloning of pineapple SLF/SFB family genes, and basis for the study of pineapple self-incompatibility mechanism.